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LECTURE 22: AXONAL GUIDANCE. REQUIRED READING: Kandel text, Chapter 54. ESTABLISHMENT OF NEURONAL IDENTITY AXONAL GUIDANCE TO TARGETS NEUROTROPHIC SUPPORT FROM TARGET TISSUES SYNAPTOGENESIS. HOW IS PROPER “WIRING” OF NERVOUS SYSTEM ACHIEVED?.

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slide1

LECTURE 22: AXONAL GUIDANCE

REQUIRED READING: Kandel text, Chapter 54

ESTABLISHMENT OF NEURONAL IDENTITY

AXONAL GUIDANCE TO TARGETS

NEUROTROPHIC SUPPORT FROM TARGET TISSUES

SYNAPTOGENESIS

slide2

HOW IS PROPER “WIRING” OF NERVOUS SYSTEM ACHIEVED?

THEORY 1: AXON PROJECTIONS ARE IMPRECISE…PROPER CONNECTIONS ARE WORKED

OUT BY ELECTRICAL ACTIVITIES DURING EARLY EXPERIENCE

THEORY 2: CHEMOSPECIFICITY MODEL….AXONS ARE DIRECTED TO THEIR PRECISE TARGETS

BY CHEMICAL SIGNALS THAT EXIST INDEPENDENT OF EXPERIENCE

Roger Sperry’s retinal axon

regeneration experiment

supported the

chemospecificity model

When optic nerve is cut, axons

regenerate and restore

normal sight and behavior

When optic nerve is cut and

the eyeball is rotated 180 deg

in socket, axons regenerate

and restore sight, but behavior

is maladaptive

FROG THINKS “UP” IS “DOWN”

And “LEFT” is “RIGHT”

slide3

Temporal

Retina

Nasal

Retina

AXON GUIDANCE IS A MULTI-STEP PROCESS

Migration towards retinal exit point (axon convergence)

Turning to exit retina and fasiculation

Fasiculated extension towards chiasm

Ipsilateral vs. contralateral migration at optic chiasm (divergence point)

Fasiculated extension into brain

Projection into lateral geniculate or into optic tectum (divergence point)

Projection a certain distance along surface of tectum (divergence step)

Diving into the tectum

Stopping at specific depths into tectum (divergence step)

Synaptogenesis

slide4

AXON DIVERGENCES AT OPTIC CHIASM ALLOW CONVERGENCE OF

CONGRUENT INFORMATION FROM BOTH RETINAS

Nasal hemiretinal axons cross over at chiasm

(contralateral projection)

Temporal hemiretinal axons do not cross over

(ipsilateral projection)

slide5

ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE

DURING ATTRACTION AND REPULSION:

GROWTH CONE STRUCTURE

ACTIN FILAMENTS

IN FILOPODIA

AND LAMELLIPODIA

MICROTUBULES

ALONG AXON AND

IN CORE OF

GROWTH CONE

slide6

ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE

DURING ATTRACTION AND REPULSION:

LAMELLIPODIA/FILOPODIA DRIVE AXON EXTENSION

slide7

ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE

DURING ATTRACTION AND REPULSION:

REPULSIVE CUES CAUSE GROWTH CONE COLLAPSE

GROWTH CONE

BEFORE

SEMAPHORIN

EXPOSURE

SAME GROWTH

CONE AFTER

SEMAPHORIN

EXPOSURE

slide8

ACTIN CYTOSKELETON DYNAMICS: THE ROLE OF RHO-FAMILY G PROTEINS

Gas, Gai, Gaq

~45,000 MW

HETEROTRIMERIC

~21,000 MW

RAS FAMILY

H-Ras, K-Ras, N-Ras

SMALL

GTPases

RhoA, Rac, Cdc42

~21,000 MW

RHO FAMILY

~21,000 MW

RAL FAMILY

RalA, RalB

GDP

GTP

ACTIVE

INACTIVE

G : GDP

G : GTP

Pi

slide9

ACTIN CYTOSKELETON DYNAMICS: THE ROLE OF RHO-FAMILY G PROTEINS

Common structure of small G proteins allows design of

constitutively active (CA) and dominant negative (DN) mutant proteins

CA-RhoA , CA-Rac , CA-Cdc42 expressed in fibroblasts induce specific architectural structures

FOCAL ADHESIONS

&

STRESS FIBERS

LAMELLIPODIA

FILOPODIA

slide11

EXTRACELLULAR AXON GUIDANCE MOLECULES ACT THROUGH RHO-FAMILY G PROTEINS

TO MODIFY GROWTH CONE ARCHITECTURE AND STEER DIRECTION OF OUTGROWTH

ATTRACTANT GUIDANCE MOLECULES

LOCAL ACTIVATION OF RAC and CDC42

LOCAL EXTENSION OF FILOPODIA and

SPREAD OF LAMELLIPEDIA

GROWTH CONE EXTENDS TOWARD

ATTRACTANT

REPULSIVE GUIDANCE MOLECULES

LOCAL ACTIVATION OF RHO

LOCAL ASSEMBLY OF ACTOMYOSIN

BRIDGES

GROWTH CONE CONTRACTS AWAY

FROM REPELLANT

slide13

TOPOGRAPHIC VISUAL MAP PROJECTED ONTO TECTUM

THE SURFACE OF TECTUM AS A TOPOLOGICALLY PRESERVED REPRESENTATION

OF THE VISUAL FIELD

AXONS FROM MOST ANTERIOR OF RETINA PROJECT TO MOST POSTERIOR TECTUM

AND VISA VERSA

WHAT IS NATURE OF THE GUIDANCE SYSTEM?

slide14

AN AXONAL REPELLENT PRODUCED BY TECTUM

POSTERIOR RETINA AXONS

WON’T GROW ALONG

POSTERIOR TECTAL MEMBRANES;

THEY GROW ALONG

ANTERIOR TECTAL MEMBRANES

OR NO MEMBRANES

ANTERIOR RETINA AXONS

SHOW NO SUBSTRATE

PREFERENCE

CONCLUSION:

POSTERIOR TECTUM PRODUCES

A CHEMOREPELLENT SPECIFIC

FOR POSTERIOR RETINA AXONS

THE CHEMOREPELLENT WAS

PURIFIED FROM TECTUM

TISSUE HOMOGENATES

REPELLENT FOUND TO BE PROTEIN

NOW TERMED EPHRIN

slide15

EPHRINS AND THEIR RECEPTORS

EPHRINS ARE CELL-SURFACE

PROTEINS:

“A-TYPE” ARE GPI-ANCHORED

“B-TYPE” ARE TRANSMEMBRANE

EPHRIN RECEPTORS ARE

RECEPTOR TYROSINE KINASES

THE SIGNALING CAPACITIES

OF EPHRIN RECEPTORS

ARE VERY DIFFERENT FROM

OTHER RTKs,

SUCH AS THE

NEUROTROPHIN RECEPTORS

slide16

GRADIENT EXPRESSION OF EPHRINS ON TECTUM

AND RECEPTORS ON RETINAL AXONS

EPHRIN EXPRESSION IS HIGHEST ON POSTERIOR TECTUM

EPH EXPRESSION IS HIGHEST ON POSTERIOR RETINAL GANGLION CELLS

AND THEIR AXONS, WHICH STOP IN ANTERIOR TECTUM

LOW EPH EXPRESSION ON ANTERIOR GANGLION CELLS ALLOWS

THEIR AXONS TO ADVANCE TO POSTERIOR TECTUM

slide17

ECTOPIC EPH EXPRESSION DIVERTS POSTERIOR RETINAL AXONS

PATCHES OF ECTOPIC EPHRIN EXPRESSION WAS ACHIEVED BY INFECTING

TECTUM WITH REPLICATION-COMPETENT VIRAL VECTOR EXPRESSING EPH

POSTERIOR RETINAL AXONS WITH HIGH EPHRIN RECEPTOR LEVELS

AVOID EPHRIN OVEREXPRESSION PATCHES

ANTERIOR RETINAL AXONS ARE IMMUNE TO EPHRIN PATCHES

(A MUTATION ELIMINATING EPHRIN EXPRESSION IN BRAIN ALLOWS

POSTERIOR RETINAL AXONS TO PROJECT THROUGHOUT TECTUM)

slide18

EPHRIN INDUCES EPH RECEPTOR CLUSTERING AND ACTIVATES THE RECEPTOR

CYTOPLASMIC DOMAIN TO INITIATE SIGNALING LEADING TO REPULSION

EPHRIN

EPH

SIGNALING

&

REPULSION

NGF

TRK-EPH

SIGNALING

&

REPULSION

slide19

CHEMOATTRACTANT PRODUCED BY FLOOR PLATE FOR COMMISURAL AXONS

COMMISURAL AXONS PROJECT VENTRALLY TO FLOOR PLATE

FLOOR PLATE EXPLANT ATTRACT COMMISURAL AXONS IN EXPLANT

OF DORSAL SPINAL CORD

slide20

IDENTITY OF FLOOR PLATE CHEMOATTRACTANT AND ITS RECEPTOR

COMMISURAL AXONS IN NETRIN AND DCC

MUTANT MICE FAIL TO PROJECT TO FLOOR PLATE

slide21

NETRIN/DCC INTERACTION CAN ALSO BE CHEMOREPULSIVE

ROLE OF CYCLIC AMP?

UNC-5 STATUS DETERMINES WHETHER DCC-POSITIVE

AXON IS ATTRACTED OR REPELLED BY NETRIN

TROCHLEAR MOTONEURONS

NORMALLY REPELLED BY NETRIN,

BUT ARTIFICIAL ACTIVATION OF

PKA IN CELLS CAUSES

ATTRACTION TO NETRIN SOURCE

slide22

CONSERVED FAMILIES OF

GUIDANCE MOLECULES

AND THEIR RECEPTORS

n

GUIDANCE

MOLECULES

GUIDANCE

RECEPTORS

slide23

AXON NAVIGATION THROUGH MULTIPLE GUIDANCE REGIONS REQUIRES

CHANGES IN GROWTH CONE SENSITIVITY TO INDIVIDUAL GUIDANCE CUES

SENSITIVITY TO ATTRACTANT IS LOST!!!

slide24

MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON GUIDANCE CUES

I. CHANGE IN INTRACELLULAR CYCLIC NUCLEOTIDE LEVELS

Early in their outgrowth, retinal axons

travel into netrin-expressing region

of optic stalk

Netrin is attractive guidance cue

Axons express high cAMP

Later in their outgrowth, retinal axons

avoid netrin-expressing deep regions

of thalamus and tectum

Netrin is repulsive guidance cue

Axons express low cAMP

Netrin mRNA

(blue)

Retinal ganglion

axons (brown)

from Shewan et al., Nature Neuroscience 5:955 (2002)

slide25

MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON GUIDANCE CUES

II. SILENCING A GUIDANCE CUE

COMMISURAL AXONS ARE

ATTRACTED TO MIDLINE FLOOR PLATE,

CROSS ONCE,

AND NEVER CROSS AGAIN

Midline floor plate produces

Netrin attractant and Slit repellent

Initially, axons are attracted by netrin and

are insensitive to slit repulsion

Upon reaching floor plate, Slit does two things:

SILENCES netrin attraction

INDUCES Slit repulsion

SLIT ACTS LOCALLY TO MODIFY

THE PROPERTIES OF THE

GROWTH CONE

slide26

SLIT SILENCES NETRIN BY GENERATING ROBO/DCC RECEPTOR COMPLEXES

AND SILENCING MUST PRECEDE SLIT-MEDIATED REPULSION

Slit induces DCC/Robo complexes,

and the cytoplasmic domain of Robo

binds the cytoplasmic domain of

DCC to block attractive signaling

The formation of DCC/Robo complexes

may also promote exocytosis of

Robo-containing vesicles,

thereby increasing surface Robo and

allowing for Slit-mediated repulsion

Only after axon passes through floor plate,

it is no longer sensitive to

floor plate attraction

Only after axon passes through floor plate,

it becomes sensitive to

Slit repulsion