Ruba Benini Pediatric Neurology (PGY-2) McGill University October 6 th, 2010 Academic Half-Day Neurophysiology 101: A humble review of basic principles

Ruba BeniniPediatric Neurology (PGY-

2)McGill UniversityOctober 6th, 2010

Academic Half-Day

Neurophysiology 101:

A humble review of

basic principles

Preamble The nervous system is a complex

organ with an intricate network of excitable cells Using transient electrical signals

for transferring information rapidly and over long distances

It is estimated that the human brain contains about 1011 neurons, with as many as 10,000 different types

Despite this complexity, the mechanisms via which neurons receive, process, generate and transmit information is essentially the similar

Neuronal signaling occurs via both electrical and chemical signals

OUTLINE

PART I : What makes nerve cells excitable? Establishment of the membrane potential Ion channels Generation of action potential and saltatory conduction

PART II: How do nerve cells communicate with each other? Synaptic transmission (central versus peripheral) Neuromuscular junction

PART III: Mechanisms of synaptic plasticity Long-term potentiation (LTP) Long-term depression (LTD) Clinical relevance

OUTLINE




Cell membrane and ion gradients

Neurons, as other cells, have a cell membrane that consists of a hydrophobic lipid bilayer that prevents/acts as a barrier to prevent diffusion of polarized molecules/ions across it

By generating ionic concentrations across the lipid bilayer, cell membranes are able to store potential energy in the form of electrochemical gradients

These electrochemical gradients are used by excitable cells such as neurons to convey electrical signals

PART I: What makes nerve cells excitable?

Membrane potential

Refers to the potential difference across the neuronal cell membrane

Resting potential is usually between -60 to -70mV with net negative inside the membrane

Membrane potential results from the separation of charge across the cell membrane Results from the unequal distribution of intracellular and extracellular ions


Membrane potential Factors contributing to the separation of charge (membrane potential) across

neuronal membranes include: Na-K ATPase pump: uses energy to set-up a concentration gradient


Membrane potential Factors contributing to the separation of charge (membrane potential) across

neuronal membranes include: Na-K ATPase : uses energy to set-up a concentration gradient K leak channels: which allow K to diffuse across the membrane along it’s

concentration gradient until it reaches equilibrium


Equilibrium potential The Nernst equation:

E = equilibrium potential, R= gas constantT= temperature in degrees Kz = charge of ion

EK: -75mV

ENa: +55mV

ECl: -60mV


The Goldman Equation: the greater the concentration and permeability of an ion, the more likely it contributes to the membrane potential

Thus, at rest, the membrane potential ( usually between -60 to – 70mV) is largely due to K+ currents

Take home point 1 Neurons store potential energy in the form of electrochemical gradients

These gradients are instituted by active/energy consuming mechanisms that maintain a high concentration of Na+ & Cl- outside the cell and high concentrations of K+ inside the cell

The voltage gradient at rest is largely due to K+ leak channels that allow K+ to flow down it’s electrochemical gradient, leaving an negatively charged inner cell membrane

Passive flux of ions (through ion channels) down their electrochemical gradient (concentration gradient & voltage across the membrane) forms the basis of electrical signaling


Ion Channels

Permeability of the cell membrane to ions is made possible by specialized transmembrane proteins called ion channels

Hydrophilic pores that allow ions to flow down their electrochemical gradient

Each channel allows > 1million ions to pass through per second thereby permitting fast transport of charged molecules

Transport across channels occurs via passive transport


Ion Channels Conduct ions: generate a large flow of ionic current

Recognize and select among specific ions

Open and close in response to specific signals (electrical, mechanical or chemical signals) – i.e. gating


Ion Channels: Voltage gated K+, Na+ and Ca2+ voltage-gated channels are structurally and genetically related


Inward current, depolarizes membrane and generates AP

Outward current, hyperpolarizes membrane

Ion Channels: Voltage gated Na+ channels have an open, inactivated and closed state


Ion Channels: Transmitter gatedPART I: What makes nerve cells excitable?

Acetylcholine receptor

Glutamatergic receptors

GABA(A) receptor

Acetylcholine

Glutamate

Serotonin

Dopamine

Glycine

GABA

Ion Channels: Transmitter gatedPART I: What makes nerve cells excitable?

GABA(B) receptor

Metabotropic Glutamate receptors (mGluR)

GABA

Glutamate

Take home point 2 Ion channels are transmembrane proteins with hydrophilic pores that, when activated, allow for

selective ions to travel down their electrochemical gradient

In general, ligand gated channels have larger pores and allow for more than one type of ion to pass through as compared to voltage-gated ion channels which are specific to single ions.

Voltage gated ion channels are involved in action potential generation

Excitatory neurotransmitters (glutamate, Ach) bind to channels that are selective for Na+, K+, Ca2+ influx of cations results in depolarization of membrane closer to threshold for action potential generation

Inhibitory neurotransmitters (GABA, glycine) bind to channels that are selective for K+, Cl- outward currents result in hypperpolarization of membrane further away from threshold for action potential generation

Metabotropic receptors mediate slower neurotransmission with longer term consequences

Mutations in ion channels clinically relevant Channelopathies: epilepsy, migraine, periodic paralysis, etc Sites of action of anticonvulsants


Axon Hillock

Generation of the Action potential Fundamental task of the neuron is to receive, conduct and transmit signals

Each neuron is continuously being bombarded by synaptic input from other neurons Apical dendrites, proximal dendrites, dendritic shaft, cell body Inputs can be excitatory, inhibitory, weak or strong


-66mV

EPSPs (Excitatory Postsynaptic Potentials)IPSPs (Inhibitory Postsynaptic Potentials)

+

_

Generation of the Action potential Voltage signal decreases in amplitude with distance from its site of initiation within a

neuron because

1. Small cross-sectional area of the cytoplasmic core of the dendrites offers significant resistance to the longitudinal flow of ions

2. inhibitory inputs at cell body can dampen signal


Generation of the Action potential

The action potential (AP) is an all or nothing response

Neuronal integration is the processes by which inputs separated by time and space are summated to reach the threshold for voltage-gated Na channels to open and thus for the AP to be generated

Temporal summation The longer the time constant,

the greater the chance for temporal summation

Spatial summation The longer the length

constant, the greater the chance for spatial summation


Generation of the Action potential The action potential (AP) is an all or nothing

response generated at the axon hillock Due to the high proportion of voltage

gated Na channels in this segment, the threshold needed to reach action potential firing is lower in this region (10mV as compared to 30mV at cell body)

Influx of Na further depolarizes the membrane thereby opening more channels which admit more Na and cause further depolarization of the membrane

This results in a rapid shift of the potential from -70mV to close to the equilibrium potential of Na of about +50mV

At this point, the net electrochemical driving force of Na+ is zero


Generation of the Action potential1. The Na+ channels open and Na+ is forced

into the cell by the electrochemical gradient causing the neuron to depolarizes

The K+ channels open slowly and K+ is forced out of the cell by its electrochemical gradient.

2. The Na+ channels inactivate at the peak of the action potential.

3. The neuron starts to repolarize.

4. The K+ channels close, but they close slowly and K+ leaks out.

5. The resting potential is overshot and the neuron falls to a -90mV (hyperpolarize)

6. After hyperpolarization the Na-K ATPase pump brings the cell membrane back to the resting potential


http://bcs.whfreeman.com/thelifewire/content/chp44/4402s.swf

Generation of the Action potential


Absolute refractory period Relative refractory period

Propagation of the Action potential – Saltatory conduction


The passive spread of the action potential down the axon occurs by electrotonic conduction This depolarization is spread by a “local circuit” current flow resulting from the

potential difference between the active and inactive regions of the axon membrane.

http://www.blackwellpublishing.com/matthews/actionp.html



The velocity of the action potential propagation is made faster by 3 main mechanisms:

Large axon diameter The larger the diameter, the lower the resistance to ionic flow

Myelination of the axons Results in a functional increase in the thickness of the axonal membrane by

as much as 100 times Acts as an insulator (↓ resistance & ↓capacitance)

Interruption of myelin: In order to boost up the signal, the axon is interrupted every 1-2mm by

nodes of Ranvier (bare patches of membrane) about 2um in length, where there is a high density of voltage-gated Na channels that can boost the amplitude of the AP and prevent it from dying out.

→ Consequently, the AP moves down the axon as though it is jumping from node to node. This is known as saltatory conduction.



The velocity of the action potential propagation is made faster by 3 main mechanisms: Large axon diameter Myelination of the axons Interruption of myelin (Nodes of Ranvier): Saltatory conduction

http://www.blackwellpublishing.com/matthews/actionp.html

Clinical Relevance: Demyelinating diseases


Waxman 1998

In diseases such as Guillame Barre syndrome, Multiple

sclerosis, peripheral axonal neuropathies, loss of the insulating myelin sheath

results in slowing of the AP conduction or complete

conduction block.

Take home point 3PART I: What makes nerve cells excitable?

The action potential is an all or none response generated at the axon hillock when neuronal integration of synaptic inputs for the cell summate to depolarize the membrane to the threshold of firing

Refractoriness of the membrane to firing immediately after an AP ensures that the signal is propagated in an anterograde fashion

The neuronal axon is enveloped by myelin sheaths from Schwann cells that are interrupted by bare segments called nodes of Ranvier where a high density of voltage gated Na channels amplifies the signal and ensures propagation of the AP towards the end of the axon terminal.

This manner of propagation of the AP along the axon is called saltatory conduction.

OUTLINE




Rapid and precise communication between neurons is made possible by 2 main signaling mechanisms: Fast axonal conduction Synaptic transmission

Synaptic transmission Electrical – gap junctions Chemical

Synapse refers to the specialized zone of contact between neurons Presynaptic and post-synaptic cell

In electrical synapses, the presynaptic and post-synaptic neurons are bridged by gap-junction channels made of Connexin proteins that conduct flow of ionic current and thus mediate electrical transmission

PART II: How do neurons communicate with each other?

Synaptic Transmission

In chemical synapses, the presynaptic and postsynaptic neurons

are separated by a synaptic cleft Transmission not as fast as electrical synapses (0.3ms to

several ms) Advantage of amplifying signal

The receptors for neurotransmitters fall into two main categories Directly ligand-gated receptors/channels:

Fast synaptic actions lasting milliseconds Role in neural circuitry that produces behavior

Metabotropic/G-protein coupled receptors:

ligand binds, activates GTP-binding protein which in term activates a channel via phosphorylation.

Slower synaptic potentials lasting seconds or minutes

Involved in strengthening synaptic connections of basic neural circuitry

Role in modulating synaptic pathways such as those involved in learning,


Synaptic Transmission

Motor unit consists of an α-Motor neuron and all the muscle fibers that it innervates One muscle fiber is innervated by only one motor neuron

The NMJ is an example of a chemical synapse where synaptic transmission is mediated by ion channels that are directly gated by Ach


Neuromuscular Junction

The motor endplate refers to the specialized region of the muscle membrane that is innervated by the motor neuron’s axon

As the motor axon approaches the end plate, it loses it myelin sheath and splits into several fine branches. A fine branch is approximately 2um thick

Each branch forms at its end multiple grape-like varicosities called synaptic boutons where the transmitter is released

At the site where the synaptic boutons lie, the surface of the muscle fiber is depressed and forms deep junctional folds lined by the basal lamina

Ach receptors are clustered at the crests of the junctional folds (10,000 receptors per um2)

Voltage gated Na channels are also clustered at the motor end-plate


Neuromuscular Junction

Stimulation of the motor neuron results in an excitatory postsynaptic potential in the muscle membrane called an endplate potential (~70mV in amplitude)


Neuromuscular Junction: Synaptic transmission

AP generated in the sarcolema travels down T-Tubules to activate voltage gated Ca2+ channels that are directly linked to Ca-channels in the sarcoplasmic reticulum

Release of Ca2+ from SR → Ca2+ bind to troponin → allows myosin-actin interaction and subsequent muscle contraction


Neuromuscular Junction: Synaptic transmission


Clinical Relevance: NMJ disorders

Lambert-Eaton

Botulism

Myasthenia Gravis

Congenital Myasthenia Gravis

OUTLINE




Synaptic Plasticity: Learning and MemoryPART III: Mechanisms of synaptic plasticity

Learning: process of acquiring new knowledge

Memory: retention or storage of that knowledge

Reflexive memory•Automatic or reflexive quality

•Not dependent on awareness, consciousness, or cognitive processes

•Occurs via slow accumulation through repetition over many trials

•Ex. Perceptual and motor skills; learning of procedures or rules

Declarative memory•Depends on conscious reflection for its acquisition and recall

•Relies on cognitive processes such as evaluation, comparison and inference

Generalizations about the neural basis of memory: Memory has stages and is continually changing (shortterm vs longterm memory) Longterm memory may involve physical/plastic changes in the brain Physical changes coding memory are localized to multiple regions throughout the CNS Reflexive and declarative memory may involve different neural systems

Reflexive → Cerebellum Declarative → Limbic structures (hippocampus)

Synaptic PlasticityPART III: Mechanisms of synaptic plasticity

In 1949, Hebb postulated that when firing in one neuron repeatedly produces firing in another neuron connected to it, changes occur in one or both of the neurons so as to strengthen the synaptic connection between them. This he postulated was the mechanism underlying learning.

Longterm Potentiation (LTP): refers to the longterm plasticity in the form of facilitated synaptic transmission induced by correlated pre- and postsynaptic activity

Longterm Depression (LTD): refers to long-lasting decrease in synaptic efficacy

EC

Longterm Potentiation

Longterm Potentiation (LTP): implicated in learning and memory formation

Hippocampus implicative in declarative memory

Majority of work looking at mechanisms for LTP have been studied in the horizontal hippocampal slice preparations in vitro 400-500um thick slices with conserved connections between dentate gyrus,

CA3/CA1 layers Slices maintained in artificial CSF Field (extracellular) and intracellular recordings can be done Stimulating electrodes

Cooke and Bliss (2006)

PART III: Mechanisms of synaptic plasticity

Perforant pathwayMossy fibers

Schaffer collaterals

EC


Delivering high-frequency trains of electrical stimuli (tetani) to Schaffer collaterals from CA3 to CA1 results in potentiation of postsynaptic response that can last for hours in vitro and for days/weeks in the intact animal

Similarly pairing a single stimulus with depolarization of the postsynaptic membrane gives a similar potentiating effect Indicating that induction of LTP is dependent on both pre- and postsynaptic activity

Nicoll et al. (1988)



EC


Nicoll et al. (1988)



Studies showed that LTP in the CA1 region of the hippocampus has 3 properties: Co-operativity (more than one fiber must be activated to obtain LTP) Associativity (the contributing fibers and the postsynaptic cell must be active together) Specificity (LTP is specific to the active pathway)

Longterm Potentiation: Molecular basis

NMDA receptors are ligand gated glutamatergic receptors that play a role in LTP induction in the hippocampus



Longterm Potentiation: Molecular basis

Activation of NMDA receptors results in influx of calcium activates Ca-signaling pathways that result in LTP induction by: Modification of postsynaptic receptors (AMPA) Activation of transcription factors that alter gene expression Changes to the number and structure of synapses Presynaptic increase in neurotransmitter release



Longterm Potentiation: Learning and Memory

Extensive evidence from in vitro and in vivo models that synaptic plasticity in the form of LTP underlies learning and memory

NMDA-R antagonism in rats impairs spatial learning Mice with mutations in NMDA receptor subunit in CA1 cells do not exhibit LTP at these synapses

and these animals have specific learning and memory deficits characteristic of hippocamal dysfunction

Evidence that cAMP-dependent pathways are need for maintenance of LTP


EC

Perforant pathway

Mossy fibers


LTP induction and maintenance varies from synapse to synapse Mossy fiber-CA3: NMDA receptors not required, nor Calcium influx, LTP mostly mediated by

enhancement of presynaptic transmitter release Perforant pathway-dentate granule cells: NMDA receptors needed, mostly postsynaptic Schaffer collaterals-CA1: same

This however is a simplification of the story – other mechanisms are sure to play a role in learning and memory

Longterm Depression (LTD)

LTD refers to activity dependent decreases in synaptic efficacy that last hours or more Occurs in several brain regions, via differing mechanisms Best studied in cerebellar cortex and hippocampus

LTD in cerebellum Induced by pairing low-frequency (1Hz) stimulation of parallel fibers (PFs) and

climbing fibers (CFs) or by pairing PF activity with direct hyperpolarization of Purkinje cells


Parallel fibers

Climbing fibersIto (2001)

Longterm Depression (LTD)- molecular mechanisms

Parallel fiber terminals → glutamate → activates AMPA and metabotropic glutamate receptors in the postsynaptic Purkinje cell.

Activation of climbing fibers → calcium enters the postsynaptic cell through voltage-gated ion channels→ ↑ intracellular calcium levels.

↑ intracellular calcium levels + DAG → activates PKC → internalization of AMPA receptors → weakening of synapse


Purves et al. (2001)

Longterm Depression (LTD)

LTD occurs in several brain regions, via differing mechanisms

Cerebellar LTD → adaptation of the vestibulo-ocular reflex

Cerebellar LTD → motor learning

Hippocampal LTD → clearing of old memories

Acute stress facilitates hippocampal LTD → stress-induced memory impairment

Visual cortex: LTD mechanisms underlie the reduced responsiveness of cortical neurons from inputs of the deprived eye

And more …


SummaryPART III: Mechanisms of synaptic plasticity

Despite the complexity of the nervous system, the mechanisms underlying neuronal signaling are essentially similar across various neuronal subtypes

Neuronal signaling occurs via electrical (changes in membrane potential, action potential generation) and chemical signals (neurotransmitters)

Ion channels form the basis of these signaling pathways

Synapses are plastic – changes are activity dependent

LTP and LTD occur in various brain regions via differing mechanisms

Despite this, in essence: LTP: enhancement in synaptic transmission involves facilitation of presynaptic

neurotransmitter release as well as postsynaptic changes (increase in receptors, structure and # of synapses)

LTD: decrease in synaptic transmission involves reduction in presynaptic release of neurotransmitters as well as postsynaptic changes (decrease in receptors, etc)

References Kandel ER, Schwartz JH and Jessell TM (1991) Principles of Neural Science. Third

Edition. Chapters 5-11, 65.

Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD (1994) Molecular Biology of the cell. Third Edition. Chapter 11.

Cooke SF and Bliss TVP (2006) Plasticity in the human central nervous system. Brain 129, 1659–1673.

Waxman SG (1998) Demyelinating diseases--new pathological insights, new therapeutic targets. N Engl J Med. 29;338(5):323-5.

Ito M (2001) Cerebellar Long-Term Depression: Characterization, Signal Transduction, and Functional Roles. Physiological Reviews, Vol. 81, No. 3, July 2001, pp. 1143-1195.

Nicoll RA, Kauer JA, Malenka RC (1998) The current excitement in long-term potentiatio. Neuron. 1(2):97-103.

Purves D, Augustine GJ, Fitzpatrick D, Katz LC, LaMantia A, McNamara JO, and Williams SM (2001) Neuroscience. Second Edition. Chapter 25.

Seizing hold of seizuresGregory L Holmes & Yezekiel Ben-Ari

Figure 1. Focal seizures result from a limited group of neurons that fire abnormally because of intrinsic or extrinsic factors.(a) In this simplified diagram, II and III represent epileptic neurons. Because of extensive cell-to-cell connections, termed 'recurrent collaterals', aberrant activity in cells II and III can fire synchronously, resulting in a prolonged depolarization of the neurons. (b) This intense depolarization of epileptic neurons is termed the paroxysmal depolarization shift. The prolonged depolarization results in action potentials and propagation of electrical discharges to other cells. The paroxysmal depolarization shift is largely dependent on glutamate excitation and activation of voltage-gated calcium and sodium channels. After the depolarization, the cell is hyperpolarized by activation of GABA receptors as well as voltage-gated potassium channels. Axons from the abnormal neurons also activate GABAergic inhibitory neurons (green) which reduce the activity in cells II and III in addition to blocking the firing of cells outside the seizure focus (cells I and IV). An electroencephalogram (EEG) recorded during this time would show a spike and a subsequent slow wave. When the balance of excitation and inhibition is further disturbed, there will be a breakdown in containment of the epileptic focus and a seizure will occur. (c) A sustained depolarization without repolarization occurs in many cells during the seizure. An EEG would show repetitive spikes during the seizure. By inducing cells to release galanin, an endogenous anticonvulsant that reduces glutamate release, Haberman et al. successfully increased inhibition and thereby reduced seizure susceptibility.

Explanation of Paroxysmal Depolarizing shift (PDS)

Documents

Ruba Benini Pediatric Neurology (PGY-2) McGill University October 6 th, 2010 Academic Half-Day Neurophysiology 101: A humble review of basic principles