Microcontrol of neuronal outgrowth

HELEN M. BUETTNER

( Nanofabrication and biosystems )

Student : Jiun-Shiung Feng Advisor : M.S. Ju

C.C. K. Lin

The Neuron

Growth cone

Neurite outgrowth is guided by the growth cone.

The growth cone consists of thin filopodia and lamellipodial veils.

http://bms.brown.edu/mppb/faculty/hkim/caption.html

Filopodial contact with remote cues can initiate rapid neurite advance to the point of contact. (O’Connor et al. 1990;Myers and Bastiani, 1993)

Proper placement of such cues in series reproduces stereotyped pathways. (Caudy and Bentley, 1986)

A model for growth cone behavior. (Buettner et al., 1994)

Mechanisms of growth cone advance (O’Connor et al. 1990)

Lamellipodial advance The forward movement of the growth cone o

ccur primarily through the net forward flow of lamellipodial veils.

Filopodial dilation (Guideposting) filopodial can detect a region of favorable su

bstrate (guidepost) and cause the growth cone to move toward it.

Microstructural paradigm Use pairs of substrates

Permissive-Nonpermissive substrate Laminin-glass (Clark et al., 1993) Laminin-albumin (Hammarback and Letournea

u,1986) Laminin-collagen (Gundersen, 1987)

Laminin ： a kind of glycoprotein, a major component of basement membranes

Figure 17.2

Quantitative framework (1/2)

Two key periods of advance

Movement across a homogeneous region according to the random walk characteristics of lamellipodial advance.

Filopodial dilation may across the nonpermissive region once filopodial contact has been made.

Quantitative framework (2/2)

is the probability that a growth cone at the border will detect and respond to a neighboring permissive region

is the number of contacting filopodia is the critical threshold

*

*

1 if

0 ifc

f fp G

f f

cp G

f*f

Experimental Methods

Instrument (Buettner, 1994) Microscope (100x objective) Video camera Videocassette(1 frame/sec ) Digitized as 8-bit computer image (256 gray l

evels) Outline is stored as binary image

Experimental Methods Lamellipodial advance is characterized by tr

acking the geometrical center of lamellipodial region.

1 1 2 2 1 1 2 2 0i i i i i iz z z a a a b

x

y

Experimental Methods

Filopodial dynamics Filopodial initiation

Initiation of filopodia appearing on a growth cone during the observation sequence (Poisson event)

Filopodial extension and retractionMeasure by plotting the trajectories of individual filopodium tips on given growth cone (filopodial length)

Experimental Methods

Filopodial length

The straight-line distance between the position of the filopodium tip at any time and its initial position

1

2 2 2

0 0i i il t x t x y t y

Experimental Results

Lamellipodial advance

The random term can be rewritten as

N(0,1) represents a random variable taken from a normal distribution with a mean of zero and a variance of 2t. (berg, 1983)

1 0i i iz z a b

1

2 0,1ia t N

12

1

12

1

0,1

0,1

i i x x

i i y

x x x t N

y y y t N

Experimental Results

Filopodial dynamicsParameters of filopodial dynamics Rate of initiation, Rates of extension and retraction, re and rr Maximun length, Lmax

Visual descriptions of growth cone motility

Quantitative growth cone response to micropatterned environment

Time to reach a border

Mean time for a neurite to track a distance L

Mean time for filopodial detection

Summary and Conclusions

Model: Probabilistic events between growth cone fil

opodia and microstructure feature. The random walk advance of growth cone.

Application: repair of nerve injuries. construction of next-generation bioartificial

organs.

Thanks for your attention