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 and during action potentials Voltage Clamp  Hodgkin and Huxley confirmed Bernstein’s hypothesis that the cellular resting membrane potential is -70mV  However, they discovered that during an AP, the voltage potential moved from -70mv not to 0 mV but towards +40mV  This was not due to a generalized breakdown of selective permeability, but rather due to…  During an action potential, there is a transient 500-fold increase in Na+ permeability followed by transient increase in K permeability (conductance)

 At rest: pK+ : pNa+ : pCl- = 10 : 0.3 : 1

 During an AP: pK+ : pNa+ : pCl- = 10 : 150 : 1  Same ionic concentrations:

[K+]out = 10mM, [K+]in = 400mM, + + [Na ]out = 460mM, [Na ]in = 50mM - - [Cl ]in = 40mM, [Cl ]out = 540 Let’s crunch numbers again

At Rest: Em= 58 x log (0.3x460 + 10x10 + 1x40) = -70mV (0.3x50 + 10x400 +1x540)

During AP: Em= 58 x log (150x460 + 10x10 + 1x40) = +44mV (150x50 + 10x400 +1x540)  That’s just the upside of the Action Potential

 What about the downside???  Following the transient opening of voltage- gated Na+ channels, they close and remain in a brief refractory state.  The shifting of the membrane potential from - 70 mV towards +44 mV results in the opening of voltage-gated K+ channels which causes the membrane to repolarize (and even hyperpolarize) its Equilibrium Potential gNa+ closes

gK+ slowly begins opening

hyperpolarization

gK+closes again

gNa+ opens Hodgkins and Huxley used the voltage clamp technique to tease apart the separate contributions of Na+ and K+ in the squid’s giant axon “What limits the size (amplitude) of the Action Potential?” The 100mV shift from -70 mV towards +40mV is based on the shift of the resting membrane potential

away from EK+ and towards ENa+

The predicted +56mV caused by the influx of Na+ ions is not quite reached as opening of K+ channels ultimately causes the action potential to reverse directions at around +40mV and repolarize.  What events limit the duration of the AP? › One factor is the refractory period of the Na+ channels, the other is the opening of K+ channels

› The refractory period is caused by a closure and relative inexcitability of voltage-gated Na+ channels following their opening

› During the absolute refractory period, these channels will not open regardless of the strength of the stimulus. This is followed by a relative refractory period where a stronger than normal stimulus can still open some Na+ channels How fast do axons conduct AP’s ? The Time Constant

T = Rm * Cm

(Rm = m. resistance, Cm = m. capacitance) › It takes time for a membrane to depolarize following an infusion of cations › In other words, the higher the membrane resistance and/or capacitance, the longer the membrane will take to build up charge and the longer it will hold a charge, resulting in a longer time for the membrane to change its voltage following a change in current How fast do axons conduct AP’s ? The Space Constant 0.5 λ = [Rm/Ri] (Rm = m. resistance and Ri = internal resistance)

 What this means is that a delivery of a pulse of cations will depolarize the membrane over a limited region of the membrane  Current delivered at point x will have maximal amplitude at x and fall off with distance How fast do axons conduct AP’s ?  Conduction speed is influenced by Axon Diameter Increased diameter drops the internal resistance to charge flow (Ri) resulting in 0.5 an increased space constant λ = [Rm/Ri]

Axonal Myelination Myelin raises Rm, which raises the ratio of Rm /Ri and increases the space constant. Myelin also reduces Cm which reduces the time constant T = Rm * Cm Saltatory Conduction  Myelinated axons have gaps between regions of myelin that are 0.5-1μm in diameter that are packed full of voltage-gated Na+ channels  These regions “re-charge” the action potential (increase the amplitude) to provide “active propagation” of the AP  The myelinated segments (“internodal regions”) can range from 50μm to over 500μm in length (depending on the diameter of the axon).

 They have reduced capacitance (T = Rm * Cm) resulting in very fast conductance speeds (1000 m/sec, smoking!)  Taken together, the AP “jumps” from node to node for a sustained AP that travels upwards towards 120 m/sec Saltatory Conduction

Nodes of Ranvier Multiple Sclerosis  MS is an autoimmune disorder resulting in CNS demyelination  CNS axonal myelination is the responsibility of oligodendroglia cells  During an MS “attack”, the patient’s own immune system damages axonal myelination initially.  This causes affected axons to not be able to transmit AP’s effectively resulting in neurological deficits, such as optic neuritis, transverse myelitis, or other focal deficits depending on the location of the lesion. Typical white matter lesions seen in patients with Multiple Sclerosis Charcot-Marie-Tooth Disease  This large group of inherited disorders is often characterized by abnormal myelination (dysmyelination) of PNS axons  Myelination of peripheral nerves is the responsibility of Schwann cells  The most common type is CMT 1A which is caused by duplications of the PMP-22 gene  Patients experience loss of vibratory sensation and weakness that begins distally in the feet and lower legs as large, myelinated peripheral nerves are affected in a length- dependent process High Arched Feet in CMT

Normal Sural Nerve Biopsy

Distal Wasting of Loss of Myelinated Leg Muscles in CMT Axons in CMT Sural Nerve The Chemical Theory of Synaptic Transmission

“How do individual neurons communicate with one another?” Henry Dale That other frog’s cousin Otto Loewi 1875-1968 1875-1968

Dale and Loewi shared the Nobel Prize in Physiology or Medicine in 1936  Dale and Loewi studied the mechanisms behind the autonomic nervous system in the 1920’s  They stimulated the Vagus nerve in a frog and watched as the heart rate slowed  They then quickly collected fluid from around the frog’s heart and injected it into the heart of another frog and found it slowed that frog’s heart rate as well, suggesting a chemical must mediate this response  They later isolated and identified the chemical as Acetylcholine a Cat Charles Sherrington 1857-1952 Nobel Prize Laureate 1932 in Medicine or Physiology  Sherrington studied spinal cord reflexes in cats  He discovered that not all neurons are excitatory in their synaptic transmission  Nerves use their pre-synaptic terminals to either stimulate or inhibit receiving cells from relaying information  Almost all inhibitory neurons are interneurons “Reciprocal Control” The idea that inhibitory interneurons bring a predictable coordinated response to a particular stimuli by inhibiting all but one of a series of competing reflexes Synaptic Potentials vs. Action Potentials  SP’s are much slower than AP’s  SP’s amplitudes can vary whereas AP’s amplitudes remain fixed  AP’s are used for long-range signaling by neurons (to send information from one region of a cell to another)  SP’s are used for local signaling between neurons (to convey information across the synapse) The Principle of Integrative Action of the Nervous System  Proposed by Charles Sherrington  At any moment, a neuron is bombarded by many synaptic signals, both excitatory and inhibitory  A nerve has only two options, to fire an AP or to not fire an AP (“…that is the question”)  It will fire an AP only if the sum of the excitatory inputs outweighs the sum of the inhibitory inputs by a critical minimum  “What is the mechanism behind this?” The Pre-Synaptic Side

“How do Action Potentials Become Synaptic Potentials?” You guessed it Bernard Katz (1911-2002)

Nobel Laureate 1970 in Medicine or Physiology  Katz studied the neuromuscular junction in frog muscles  He showed that Acetylcholine released from motor neurons accounts for all phases of the synaptic potential  Furthermore, he showed that neurotransmitters are released from axon terminals not as individual molecules, but rather from small packets known as “quanta” that contain about 5,000 molecules each That other squid’s sister Bernard Katz (1911-2002) Nobel Laureate 1970 in Medicine or Physiology  Using the synapse of the squid’s giant axon, Katz showed that as an AP moves into the pre-synaptic terminal, it leads to the opening of voltage-gated Ca++ channels that admit Ca++ ions into the cell.  The influx of Ca++ ions lead to the fusion of synaptic vesicles to the surface membrane of the pre-synaptic terminal  This results in the release of the contents of the synaptic vesicle (a quanta of neurotransmitter) into the synaptic cleft  Up to 200 synaptic vesicles full of neurotransmitters are released into the synaptic cleft for each action potential that reaches the terminal zone of a neuron

Botulism  Botulism is caused by the botulinum toxin produced by Clostridia botulinum  The toxin binds to the membrane surface of acetylcholine axon terminals and it’s light chain is taken up into the cytoplasm by endocytosis  The light chain of botulinum toxin type A then causes proteolytic cleavage of the SNAP-25 protein which is responsible for the fusion of the synaptic vesicles containing acetylcholine to the axon terminal membrane  The result is skeletal muscle paralysis and parasympathetic nervous system failure (both rely on the release of acetylcholine) Botulism

Infantile Botulism Wound Botulism

Systemic Botulism Botox  A released neurotransmitter has a good shot at binding to a post-synaptic receptor and is soon released.  NT’s must then be either enzymatically degraded within the synaptic cleft or recycled (re-uptake) into the pre-synaptic terminal or into adjacent glial cells  This allows for punctate signaling which is essential for the "integrative” quality of nervous system signaling Organophosphate Poisoning  Certain pesticides contain chemicals that interfere with the functioning of Acetlycholinesterase whose function is to help clear the synaptic cleft of ACh  Nicotinic ACh effects: › Weakness, Fasiculations, Cramping  Muscarinic Ach effects: › “DUMBELS” › Diaphoresis/Diarrhea, Urination, Miosis, Bradycardia/Bronchospasm, Emesis, Lacrimation, Salivation The Post-Synaptic Membrane Sherrington’s Cat John Eccles (1903-1997) 1963 Nobel Laureate in Physiology or Medicine  Studying the spinal reflex in cats, Eccles confirmed Sherrington’s conclusions that motor neurons receive both excitatory and inhibitory inputs  He found that when motor neuron dendrites are exposed to excitatory neurotransmitters, the resting Membrane Potential depolarizes from -70mV to -55mV, which is the threshold potential for firing an AP  He also discovered that when the same neurons are exposed to Glycine, the Membrane Potential becomes hyperpolarized from -70mV to -75mV, making it more difficult for the cell to fire an AP Electrical Synapses  (Let’s get these suckers out of the way)  Initially felt to be rare, these synapses are becoming increasing recognized  Provide an efficient transfer of an AP from one neuron to another  The physiological role of electrical synapse is currently being worked out Chemical Synapses  Much more common than electrical synapses  Both types of Chemical Synapses result in EPSP or IPSP  Ionotropic (direct) Receptors › Coupled to membrane-spanning ion channels (ionophores) › Duration of effect is very quick (few msec)  Metabotropic (indirect) Receptors › Alters the phosphorylation state of G-coupled proteins › Results in local changes in efficacy of ionophores, which can then change the membrane potential › Duration of effect fast (10-20 msec) or long (up to minutes) IONOTROPIC METABOTROPIC

direct, simple, fast (0.2 msec) Indirect, g-protein coupled, not fast (but hardly slow…3-15 msec) Chemical Synapses  End result of both types of receptors is to either increase (EPSP) or decrease (IPSP) probability of the post-synaptic cell firing an AP by either depolarizing or hyperpolarizing its membrane potential  This change in membrane potential is itself determined by the particular Reversal Potential for each neurotransmitter receptor  The Reversal Potential is determined by which ions are let into or out of the cell through the ion channels associated with each chemical receptor. The Reversal Potential is the point where there is no longer any net flow of ions. Flow of ions is determined by each individual ion’s specific Nernst Potential Nicotinic Acetylcholine Receptor  Found on the muscle membrane side of the neuromuscular junction in skeletal muscle  Binds to Ach released from α-Motor neurons  Requires 2 ACh molecules to bind to open ionophore  Activations opens an ionophore that allows inward movement of Na+ ions and outward flow of K+ ions  Since the driving force for Na+ ions is so much greater than K+ ions, the net result is depolarization of the

membrane towards VNa+ (+56 mV) for a resulting membrane Reversal Potential ~ -5mV Myasthenia Gravis  An auto-immune disorder with antibodies binding to Ach receptors in the NMJ  Causes fatigable weakness, ptosis, and ophthalmoparesis  Can be diagnosed by observing a transient increase in strength following intravenous administration of Tensilon (an acetylcholinesterase inhibitor which transiently increases the amount of Ach available to the reduced number of Ach receptors) Ptosis

Electrodecremental CMAP Response NMDA Glutamine Receptor  Ionotropic receptor  Glutamate is the most common excitatory NT in the CNS  Glutamate binding to receptors causes EPSP’s in post- synaptic neurons  Its ionophore is a mixed Na+ /K+/Ca++ which has a Mg+ ion blocking the entry (or egress) of any ions until the membrane becomes depolarized (usually by Glutamate binding to a sister receptor, AMPA)  Once Mg+ is out of the way, Na+ and Ca++ flow in and K+ flows out for a Reversal Potential of ~ -10mV (with the largest contributor coming from Na+ ) NMDA Receptor Anti-NMDA Receptor Encephalitis  Presents with fever, headache, and signs of delirium  Progresses towards seizures, central hypoventilation, autonomic instability and akinetic mutism, often followed by facial dyskinesias  CSF shows a low grade pleocytosis and MRI may be normal or show increased T2 signal in the medial temporal lobes  Positive IgG or IgM antibodies to the NR2 subunit of NMDA Receptors found in serum and CSF  May respond to glucocorticosteroids or IVIg  Associated with ovarian teratomas as a paraneoplastic syndrome Anti-NMDA Receptor Encephalitis

Rat brain hippocampus stained with anti-IgG anti- bodies to the NMDA receptor from affected FLAIR MRI showing patient increased signal in hippocampi GABAA Receptor  Ionotropic receptor  Duration of effect is quick (10-15 msec)  Activation leads to opening of Cl- channel  Reversal Potential is the same as the Nernst potential - for Cl (VCl-~ -71mV)  GABA is the most common inhibitory NT in the CNS  GABA binding to its receptors causes a short circuit of EPSP’s generated by other receptors GABAB Receptor  Metabotropic Receptor  Duration of effect is much longer (100 – 200 msec)  Activation of the receptor causes activation of linked G-Proteins that in turn result in the opening of K+ channels  Reversal Potential is therefore nearly the same as + the Nernst Potential for K (VK+~ -90mV) GABAAReceptor  Agonist: GABA  Antagonist: Bicucuuline  Positive Allosteric Modulators: Benzodiazepines, Barbiturates, Ethanol, Propofol, Inhalation Anesthetics  Negative Allosteric Modulators: Flumazenil

GABAB Receptor  Agonists: GABA, Baclofen, GHB (gamma-Hydroxybutyric Acid)