Ted Weyand, PhD Stephen Deputy, MD November, 2009 November, 2009 June, 2010 Cajal’s Neuron Doctrine The Ionic Hypothesis The Chemical Theory of Synaptic Transmission a Rat a Rabbit Santiago Ramón y Cajal (1852-1934) 1906 Nobel Laureate in Physiology or Medicine (with Camillo Golgi) 1. The neuron is the fundamental structural and functional element of the brain 2. The terminals of one axon communicate with the dendrites of another only at specific sites 3. The principle of Connection Specificity 4. The theory of Dynamic Polarization 1. The neuron is the fundamental structural and functional element of the brain H and E stain of the Cortex H and E stain of Cortical Neurons Drawings by Cajal of silver-impregnated neurons using a stain devised by Camillo Golgi 2. Cajal inferred that the terminals of one axon communicate with the dendrites of another only at specialized sites (synapses). He further inferred that there must be a small gap (synaptic cleft) where the axon of one neuron communicates with the dendrites of another. 3. The Principle of Connection Specificity Each nerve cell forms synapses and communicates with certain nerve cells but not with others. Most neurons make contact with the dendrites of many target cells. Conversely, the dendrites of a target neuron receives signals from many pre- synaptic neurons. Nerve cells are thus linked in specific pathways referred to as neural circuits and signals travel along these pathways in a predictable pattern. The human brain contains Approximately 100 Billion neurons With about 1,000 synaptic connect- ions each. This results in approximately 1,000,000,000,000,000 (one quadrillion) synaptic connections. 4. The Theory of Dynamic Polarization Signals from a neuron travel in only one direction. “Information” flows from the dendrites along the axon to the pre-synaptic terminals. From there, it moves across the synaptic cleft to the dendrites of the next cell and so on. So what is this “information” anyways? Describes the mechanisms in which individual nerve cells generate electrical signals (action potentials). “How do we know this stuff???” With a little help from our scientific and animal friends. Luigi Galvani A Frog 1737-1798 In 1791, Galvani left a He proposed that frog leg hanging from a nerve and muscle cells copper hook from his are capable of iron balcony. generating a flow of The resulting electrical electrical current. flow from two dissimilar metals resulted in the frog leg kicking. Hermann von Helmholtz 1821-1894 Another Frog In 1859, Helmholtz discovered that electrical signals are actively propagated down axons at speeds of around 90 feet per second (for large myelinated axons) This is much slower than electricity moves across a wire (186,000 miles per second) Unlike wire, the electrical signal in axons does not diminish with distance Edgar, Lord Adrian 1889-1977 Nobel Laureate 1932 A Different Frog in Medicine or Physiology (with Charles Sherrington) Adrian placed a piece of metal on the outside of a sensory axon signaling from a stretch receptor in frog leg muscle and recorded action potentials He discovered that: AP’s consistently last 1/1000 of a second in duration. Each AP has the same duration and amplitude (shape) regardless of the intensity of stimulus. More intense stimuli result in an increased rate of firing of AP’s compared to milder stimuli (a light stimulation results in 3 AP’s/sec vs. a painful stimulation which fires 100’s of AP’s/sec). “What is the Mechanism that Underlies the Generation and Propagation of Action Potentials?” The Membrane Hypothesis The Resting Membrane Potential The Action Potential The Membrane Hypothesis The Resting Membrane Potential The Action Potential Julius Bernstein The Second Frog’s 1839-1917 Twin Brother (a student of Helmholtz) The Membrane Hypothesis Cytoplasm and extracellular fluid does not contain free electrons (like metals), but rather electrically charged atoms (ions) such as Na+, K+ and Cl- that can carry current. Membranes must be able to separate charges (ions) to create a voltage potential. The Membrane Hypothesis The Resting Membrane Potential The Action Potential Extracellular Fluid Lots of positively charged Na+ ions balanced by negatively charged Cl- ions Cytoplasm Fluid Positively charged K+ ions balanced by negatively charged proteins The positive and negative charges on either side of the membrane are balanced, but with different ions used Even at rest, an axon exists at a steady potential which is approximately -70mV (inside of cell more negative compared to outside). This is the resting membrane potential. In the resting state, Bernstein concluded that the membrane is impermeable to all ions except for K+ (good guess, close). The Nernst Potential Equation E = RT/ZF x log ([ionout]/[ionin]) E=Equilibrium potential, R=gas constant, T=temp (K), Z=valence of ion, F= Farraday’s constant. At 37° (C), E=58 log ([ionout]/[ionin]) The Nernst Potential Equation E = RT/ZF x log ([ionout]/[ionin]) “Why is this equation important anyways?” Because it helps to predict the resting Membrane Potential (Equilibrium Potential, Nernst Potential) based on single ions that have been separated in concentration by a barrier (cell membrane here) The K+ ion + + [K out] = 10mM and [K in ]= 400mM (thanks in part to the Na/K ATPase pump) E = RT/ZF x log ([ionout]/[ionin]) 0 EK= 58 log (10/400) = -90mV (at 37 C) The K+ ion K+, like all ions, has two taskmasters, entropy (which drives it down it’s concentration gradient) and quantum law (which accounts for charge attraction) The effect of charge on the electrochemical gradient is much larger than the effect of concentration. Small changes in concentration result in large changes in electrical potential The membrane, selectively permeable to K+, but not Cl- (and others) K+ moves down its concentration gradient, but braked by its electrical gradient Initial 0 mV Equilibrium ~ - 90 mV A separation of charge (potential) now exists across the membrane But wait!!!!! “You said the cellular resting membrane potential was -70mV and not -90mV as predicted by K+ using the Nernst equation! What is going on then?” While K+ is the main player, the cellular membrane is also somewhat permeable to other ions, such as Na+ and Cl- Here, we need to understand the role of the Na+/K+ATPase pump and the Goldman Equation Na+/K+ ATPase pump Opposite to K+, Na+ concentrations are much higher on the outside than on the inside This is caused by the Na+/K+ ATPase pump which kicks out 3 Na+ ions for every 2 K+ ions that it drives into the cytosol The pump requires ATP to drive the ions against their concentration gradients. Na+ (that other ion) The Nernst Potential Equation for Na+ based on concentrations of + + [Na ]out = 460 mM and [Na ]in = 50 mM predicts an Equilibrium Potential of… 0 ENa= 58 log (460/50) = +56mV at 37 (C) While Na+ and K+ have different Membrane voltage potentials (as determined by the Nernst Equation), they also have very different membrane permeabilities which will contribute to the ultimate resting membrane potential of all cells. At rest, K+ ions flow more freely through passive K+ channels, whereas Na+ ions are relatively restricted in their flow across membranes. Hence, the resting membrane potential is closer to EK+ than to ENa+ What about Cl- ? Cl- is intermediate in permeability between K+ (high permeability) and Na+ (low permeability) in the resting membrane state. - However, the Cl membrane potential, ECl- (-71mV), is close to that of the cellular resting potential (-70mv) so it does not play much of a role in the resting state. In other words, opening more Cl- channels won’t change the resting membrane potential by much but can short circuit changes in membrane potential caused by the influx of other ions (Na+ , K+, Ca++) Takes into account both concentration gradients as well as partial permeabilities of individual ions across a membrane to predict the actual membrane potential at rest or during an action potential Here, P= The partial permeability of the ion in question (you know the rest all ready) Let’s Crunch Some Numbers Assuming Pk/Pna/PCl = 10/0.3/1 and + + [Na ]out=460mM, [Na ]in=50mM, [K+]out=10mM, - - [K+]in=400mM, [Cl ]in=40mM, [Cl ]out=540 Em= 58 x log (0.3x460 + 10x10 + 1x40) (0.3x50 + 10x400 +1x540) Em = -70 mV (Voilá) Now that we understand the neurophysiological principles that determine the cellular Resting Membrane Potential, It is time to ask… What about the Action Potential??? The Action Potential is truly amazing! During the AP, the local membrane potential suddenly moves from -70mV to +40mV in 1/1000 of a second. “How does this happen?” As mentioned before, Julius Bernstein applied positive ions from a battery to a frog leg axon. He predicted that during an AP, the selective permeability of the membrane breaks down transiently allowing free passage of all ions. This would cause the membrane potential to move from -70mV to 0mv. Bernstein’s hypothesis of the breakdown of selective ionic permeability causing AP’s was challenged and ultimately disproven by Hodgkin and Huxley in the 1930’s Alan Hodgkin* Andrew Huxley* 1914-1998 A squid b.1917 * Both shared the Nobel Prize in Medicine or Physiology in 1963 Hodgkin and Huxley used the giant axon of the squid (50x the wider than any axon in humans) They inserted a pipette tip into an axon and another into the extracellular fluid to form a voltage clamp This way, they could study the movements of specific ions into and out of the axon at rest