Ted Weyand, PhDStephen Deputy, MD

November, 2009

November, 2009

June, 2010

Cajal’s Neuron Doctrine

The Ionic Hypothesis

The Chemical Theory of Synaptic Transmission

1906 Nobel Laureate in Physiology or Medicine(with Camillo Golgi)

Santiago Ramón y Cajal(1852-1934)

a Rat a Rabbit

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 Specificity4. 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 containsApproximately 100 Billion neuronsWith about 1,000 synaptic connect-

ions each.

This results in approximately1,000,000,000,000,000

(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 Galvani1737-1798

A Frog

In 1791, Galvani left a frog leg hanging from a copper hook from his iron balcony.

The resulting electrical flow from two dissimilar metals resulted in the frog leg kicking.

He proposed that nerve and muscle cells are capable of generating a flow of electrical current.

Hermann von Helmholtz1821-1894 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 Adrian1889-1977

Nobel Laureate 1932in Medicine or Physiology(with Charles Sherrington)

A Different Frog

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 Bernstein1839-1917

(a student of Helmholtz)

The Second Frog’s Twin Brother

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 toseparate 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])

EK= 58 log (10/400) = -90mV (at 370 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)

Initial 0 mV Equilibrium ~ - 90 mV

K+ moves down its concentration gradient, but braked byits electrical gradient

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 pumpOpposite to K+, Na+ concentrations are much higher on the outside than on the insideThis is caused by the Na+/K+ ATPase pump which kicks out 3 Na+ ions for every 2 K+ ions that it drives into the cytosolThe pump requires ATPto drive the ions against theirconcentration 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…

ENa= 58 log (460/50) = +56mV at 370 (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 principlesthat 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*1914-1998

Andrew Huxley*b.1917

* Both shared the Nobel Prize in Medicine or Physiology in 1963

A squid

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 notto 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+ opens

gNa+ closes

gK+closes again

hyperpolarization

gK+ slowly begins opening

Hodgkins and Huxley used the voltage clamp techniqueto 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

λ = [Rm/Ri]0.5

(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 byAxon Diameter Increased diameter drops the

internal resistance to charge flow (Ri) resulting in an increased space constant λ = [Rm/Ri]0.5

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

Nodes of Ranvier

SaltatoryConduction

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 matterlesions 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 ArchedFeet in CMT

Normal Sural NerveBiopsy

Loss of MyelinatedAxons in CMT Sural

Nerve

Distal Wasting of Leg Muscles in CMT

The Chemical Theory of Synaptic Transmission

“How do individual neurons communicate with one another?”

Henry Dale1875-1968

Otto Loewi1875-1968

Dale and Loewi shared the Nobel Prize in Physiology or Medicine in 1936

That other frog’s cousin

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

Charles Sherrington1857-1952

Nobel Prize Laureate 1932 in Medicine or Physiology

a Cat

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?”

Bernard Katz(1911-2002)

Nobel Laureate 1970 in Medicine or Physiology

You guessed it

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

Bernard Katz(1911-2002)

Nobel Laureate 1970 in Medicine or Physiology

That other squid’s sister

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

Systemic Botulism

Wound 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

John Eccles(1903-1997)

1963 Nobel Laureate in Physiology or Medicine

Sherrington’s Cat

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+ )

NMDAReceptor

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

FLAIR MRI showingincreased signal inhippocampi

Rat brain hippocampus stained with

anti-IgG anti-bodies to theNMDA receptorfrom affectedpatient

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)