BIOL 2480 - Constructing Neural Circuits

Hindbrain pattern formed by differential

Hox gene expression

FGF-8 determines orientation for

hindbrain

WNT determines

midbrain fate

center-surround - neuron surrounded by

non-neurons, the neuron is the 1st neuroblast to bind notch

Neurons migrate with

adhesion molecules, NCAMs

Many cells have an

apical side and a basal side

Apical side interacts with

environment,

basal side secretes

materials

Neurons are a specialized version of this, with dendrites representing

apical side

and axons representing

•basal side.

1st step of differentiation is neuroblast becoming a

polarized neurite

Neurites are neural fated cells that begin to send out extensions but do not yet have an

identifiable dendritic or axonal field

Once a neurite starts to expand, organelles and cytoskeleton are transported to form

axons and dendrites

Tubulin forms long

parallel microtubules

Actin forms

microfilaments

Motor proteins associated with microtubules traffic

cell products to axons and dendrites

Polymerization requires

ATP

Anterograde: from

nucleus to terminus

Retrograde: from

terminal to nucleus

After neurite formation, transport forms

axonal and dendritic branches

Polymerization of both tubulin and actin relies on ATP so is an

active process

PAR proteins are differentially expressed in axonal sectons and push microfilaments; disrupting PAR signalling stops

axon formation

Protein and other cellular trafficking relies on microtubular highways to

transport materials (cargo).

Must traffic all the synaptic proteins we talked about last time as well as some

NT vesicles as well as mRNA needed to synthesis proteins at terminal.

How do axons extend? Send out a

•growth cone as 1st explorer.

Lamelipodia -

main cone

Filopodia-

small extensions

actin is concentrated on

outside, in filopodia

Tubulin is in

middle, in lamellipodium

•Send out a growth cone as 1st explorer.

Actin filaments push

filopodia, microtubules push lamellipodia

Microfilaments are formed when G-actin (globular) assembles into

F-actin (filamentous)

New F-actin gets made in leading edge and broken down back in

lamellipodium

Axon extends forward as growth cone like and amoeba. The growth cone is a big bulb with small side extensions off of it. The small extensions are the filopodia

Microtubules extend into central core and extended cytoplasm collapses to make

new axon segment.

Actin attaches to integral proteins - links to

substrate and pulls along

Attractive cues lead to

actin polymerization

repulsive cues lead to

actin depolymerization

How do axons know where to go? 6 basic ways

1.Extracellular matrix adhesion

2.Cell surface adhesion

3.Fasciculation

4.Chemoattraction

5.Contact inhibition

6.Chemorepulsion

A given axon can use all or some of these mechanisms for

axon guidance

Diffusible factors (4&6) only work at close distance but don't require

actual binding to another cell.

Growth cone is a sensory and motor structure that responds to guidance cues by "transducing positive and negative cues into signals that regulate cytoskeleton and thereby determine

course and rate of axon outgrowth."

Receptors on filopodia do not just mediate adhesion but also are

signal-transducers coupled to various second messengers that affect cytoskeletal components to affect growth cone direction and rate of growth.

Extracellular matrix adhesion- The extracellular matrix (ECM) is full of adhesion molecules like

laminins, collagens and fibronectin

The ECM can then lay down a pathway to guide growth cones along a

defined path

If you put growing axons on a petri plate with "collagen-coated corridors" they will grow along the corridors in absence of other cues and avoid

non-adhesive surfaces

Laminins are major components of all

basal laminae (aka basement membrane - ECF around muscles and epithelia) in both verts and inverts

Integrins are transmembrane receptors in

developing growth cone

Growth cone integrins interact with

ECM proteins

Integrin binding sets off signal cascade that encourages

actin polymerization

Integrins are integral membrane proteins found in all cells with so a variety of heterodimers. Each dimer specifically binds to different sorts of ECF and ECF components also differ (at least 7 different laminins) so you can get specific pathway guidance just based on the

particular combo of integrins and ECF components.

Once bound the integrins set off signaling cascades that ultimately results in movements of actins to drag

filopodia along. (Ca2+ probably plays a role here)

Cell adhesion molecules stick cells together but can also promote

neurite outgrowth

Cell adhesion molecules •Two classes:

I:

-cadherins - need Ca2+

Binding activates catenins, which affect

actin assembly

MANY different types of cadherins and they prefer to bind to their own type so

VERY specific

Cadherins are found in cells throughout the body and the nervous system is full of N-cadherins (as well as other cadherin types). Cadherins on adjacent cells interact to form adhesive bonds, with each prefering to bond to its own kind (over 100 different types). This allows for very specific interactions.

In cadherins, intercellular domain interacts with proteins called

catenins that then affect organization of actin cytoskeleton

Get active polymerization of actin components when the 2

catenins touch

•Class II:

-CAMs - Ca2+ independent

-CAMs - Ca2+ independent

More indirect effects than

cadherins

CAM binding activates kinase pathways to influence

growth cone

CAMs are part of a diverse immunoglobulin superfamily. CAMS may want to bind to its own kind but

CAMs can also bind to many other things as well).

For both types initial extracellular adhesion leads to cytoplasmic reactions that strengthen the

connection

CAMs are still diverse in intracellular activity. Intracellular domain sets off variety of signalling pathways that have more

indirect effects but still direct neurite outgrowth.

Fasciculation means bundling of

axons together along a pathway.

A "pioneer" axon leads the way and then

subsequent ones will bind to it and grow along it.

what mediates fasciculation

NCAMs, same neural type, encourages growth from like neurons

Chemoattraction- Axons can grow up or down a concentration gradient to be guided in a

•specific direction (tropism).

Tropic factors are

attractive

trophic factors support

survival

Best known tropic factor are

netrins

Netrin is released by signalling cell and binds to receptor

(DCC/UNC5) on growth cone

Receptor activation "attracts"

cytoskeleton

between tropHic (promote

neural survival)

tropic factors (promote

directional outgrowth)

Netrin is released by signalling cell and binds to receptors (DCC, UNC5, others) on growth cone. This causes

cytoskeletal rearrangements to go toward the source.

More netrin you have, the more activation of DCC you have and the more growth you get in that region.

Netrins are especially prevalent in

floorplate/midline of developing nervous system.

Once axon crosses midline it doesn't want to go back so it looks like slit changes response of neuron to netrin so it won't go back over midline once it is

crossed

Contact inhibition- Both semaphorins and ephrins can stop growth via

growth cone collapse. This may cause direction change

1) Some semaphorins are membrane bound. They bind to Plexin receptors in neural cell membrane and cause actin to fall apart. This stops filopodia from

extending out

2) Growth is concentrated elsewhere. As soon as growth cone touches a cell with semaphorins it will stop

growing in that direction. Ephrins work the same way, just different receptors.

Semaphorins can also act as diffusible factors, in which case they bind to

neuropilins but same effect is seen.

Chemorepulsion- Diffusible factors can make an axon turn

•away from an area.

Two major ones are

semaphorins and netrins.

•But wait, you just said netrins are chemoattractants! Which is it?

depends on

receptors and second messengers

Presence of Slit&Robo cause

growth cone collapse

slit + netrin = inactivation of PKA, follows guidance molecules away from

midline

If a neuron has DCC receptors it will turn towards

netrin

If it has UNC-40 homologs it will

turn away

Second messenger pathways mediate responses. It is thought that this happens at

midline DRAW

•axon guidance in the visual system

Ganglion cells have to leave the retina, enter the

optic tract, and find the appropriate target, some crossing the brain and some not.

1) uses chemoattractants to determine

out

2) ECM to keep growing in

that direction

3) Fasciulation (NCAMs telling

where to go)

4a) content mediated attraction (in chiasma), some have

receptors and some don't (determines left from right)

4b) follow

chemical guidance cues

5) in tectum, follow

radial glia

6) stop at

synapse

Okay, they got there but how do they form synapses

Synaptogenesis

•Much of what we know is based on the neuromuscular junction

•5 basic steps

1. growth cone approach

2. unspecialized contact

3. synaptic vesicles accumulate, basal lamina forms

4. multiple axons converge

5. only one survives

1. Growth cone approaching a newly formed filament. Growth cone undifferentiated but guided by things we just finished talking about. As it approaches it has some capacity for

neurotransmitter release but it is unspecialized

2. 1st point of contact is essentially random within the approach zone. Contact is functional but very simple with little

morphological specialization