Construction of Neural Circuits

Question 1

What are polarized epithelial cells?

Answer 1

cells that will develop into neurons

These all have an apical side specialized for secretion of chemical signals and a basal side involved in receiving intercellular signals

The distribution of cytoskeletal proteins creates this apical/basal polarity

Question 2

What do fully committed polarized epithelial cells extend

Answer 2

Once a cell has fully committed to the neural fate, it begins to extend numerous, temporary outgrowths called neurites

Eventually, actin and microtubule elements will redistribute and concentrate in a single neurite that will be the axon. The remaining neurites will become the dendrites.

Question 3

Two components of the axon growth cone

Answer 3

A structure at the tip of the growing/developing axon that guides it to its’ appropriate target.

Lamellipodium – the larger, sheet-like component of the growth cone (the “cone” part)

Filopodia – the smaller, finger-like projections that extend out from the lamellipodium

Question 4

What is the growth cone for?

Answer 4

The growth cone is capable of detecting signals that guide the axon to its appropriate target. This includes changes in direction when necessary (think about how the axons of the retinal ganglion cells are distributed in the optic chiasm/optic tract).

Once the appropriate target is reached the growth cone converts into a presynaptic terminal.

Question 5

What is the movement of the growth cone controlled by? How does the axon grow to follow the growth cone?

Answer 5

Movement of the growth cone and the axon shaft is controlled by the assembly and disassembly of the cytoskeletal proteins.

Actin forms the filaments that create the structure of the lamellipodium and filopodia.

Tubulin forms the microtubules that create the structure/shape of the axon. Microtubules are also responsible for the trafficking of proteins, mRNAs, vesicles and other cellular components up and down the axon.

Question 6

What happens in the growth cone during projection toward a cue?

Answer 6

When the growth cone is projecting towards a cue, globular or monomeric actin (g-actin) is polymerized to form fibrillar actin (f-actin) that forms the actual actin filaments. Happening at the tip or leading edge.

This process pushes the filopodium forward (the actin filaments are connected to membrane-bound proteins to provide the necessary mechanical stability).

Other regions of the growth cone are undergoing actin fibrile depolymerization.

A similar process is occurring with tubulin; tubulin monomers are polymerized to form microtubules at the end closest to the growth cone, extending the axon.

Question 7

What is the polymerization of actin/ tubulin in the growth cone controlled by?

Answer 7

The process of polymerization of actin and tubulin is controlled by actin-binding and tubulin-binding proteins.

The activity of these assembly proteins is regulated by intracellular Ca2+. The sources for this Ca2+ signal include:

Voltage-gated Ca2+ channels

TRP channels (why?)

Ca2+ release from intracellular stores

Thus, Ca2+ signals are a critical component for determining the direction/path of the growth cone.

Question 8

What does the axon cone eventually become?

Answer 8

the presynaptic terminal

Question 9

What are the types of non-diffusing axon guidance cues?

Answer 9

extracellular matrix adhesion molecules (ECMs)

cell adhesion molecules (CAMs) and Cadherins

ephrin-eph complexes

Question 10

Describe how the extracellular matrix adhesion (ECMs) molecules guide axonal growth

Answer 10

These are proteins such as laminin, collagen & fibronectin that are secreted by the cell and then polymerize to create a complex that remains in close proximity to its source.

The receptor for these ECM molecules are integrins.

When a cell/axon expressing integrins make contact with ECM molecules, the integrins initiate an intracellular signaling cascade that regulates the path of axon growth.

Usually used for growth along tissue borders, e.g. the neural tube and the surrounding mesenchyme.

Question 11

describe how the cell adhesion molecules (CAMs) and cadherins guide axonal growth.

Answer 11

CAMs and Cadherins:

Expressed on both the growing axon and the target tissue.

These undergo homophilic binding, so that whenever two of the same protein encounter each other, they bind and initiate an intracellular signaling cascade.

CAMs are associated with fasiculation, the groups of axons as part of a nerve.

Cadherins play a role in axonal target selection and the conversion from growth cone to functioning synapse.

Question 12

Describe how the Ephrin-Eph complex guides axonal growth.

Answer 12

Ephrin is technically the ligand in this type of signaling, despite the fact that it is a membrane-bound protein.

Eph is the receptor. It is a tyrosine kinase receptor (see Chapter 7)

The signaling is bi-directional. Ephrin-Eph binding can initiate signaling cascade within the ephrin-containing cell via activation of protein kinases. A signaling cascade can also be initiated in the eph-containing cell via the tyrosine kinase mediated process.

This pathway can also be used to limit axon growth by the cleavage of either ephrin or eph.

Produce a repulsive signal. Both cells have signalling cascades

Question 13

What are axon guidance diffusible signals?

Answer 13

These are secreted molecules that diffuse to form a signaling gradient that can either be attractive or repulsive.

One issue in identifying these molecules was distinguishing between those responsible for guidance (tropic) vs. those responsible support/survivial/growth (trophic).

Many of these molecules or signaling complexes were initially identified in C. elegans or Drosophila

*May help thing to pass through the substance. *

Question 14

Netrins

Answer 14

secreted protein that is chemoattractive when it binds to the DCC receptor and chemorepellant when it binds to the Unc5 receptor.

Both receptors interact with Rho/Gap signaling cascades that act on axonal cytoskeletal elements.

Question 15

Slit/Robo

Answer 15

slit is the secreted protein and robo is its’ receptor. Are chemorepulsive

and also work through the Rho/Gap pathway

Question 16

Semaphorins

Answer 16

chemorepulsive factor (can be an attractant too); is actually non-diffusible (bound to cell membranes or ECM). Its receptors are plexin and neuropilin (which interacts with the Rho/Gap pathway).

Question 17

Describe the example of axonal guidance involving the floor plate.

Answer 17

As the neural tube develops into the spinal cord, axon that will form the spinothalamic tract project to the floorplate that express high levels of netrins.

Netrin attracts the axons until they cross the ventral midline (axons express DCC receptors)

Once the axons cross the midline, 2 factors prevent them from re-crossing

1. Expression of the robo receptor which makes the axons now sensitive to the chemorepulsive signal from slit.
2. Axon’s lose their sensitivity to netrins (DCC receptors not-expressed)

Mutations of the netrin gene disrupt the formation of this midline-crossing axonal pathway.

Question 18

What is the initial stage of synaptogenesis

Answer 18

Initial stages mediated by the same cell-adhesion molecules used for axon-guidance: cadherins, CAMs & ephrins.

Question 19

What is the second stage of synaptogenesis (general, including protein names)

Answer 19

, the two cells undergo specialization to establish their presynaptic & postsynaptic identities.

Neuregulin

neurexin/neuroligin

Question 20

What does neuregulin do in synaptogenesis?

Answer 20

transmembrane protein expressed on the presynaptic neuron; extracellular region is cleaved and it diffuses across the cleft to bind to the ErB receptor (a receptor kinase); this interaction contributes to the synthesis and insertion of NT receptors

• expressed presynaptically, works postsynaptically.*
• leads to addition of post-syn receptors*

Question 21

What does neurexin/neuroligin do in synaptogenesis

Answer 21

Neurexin/Neuroligin – adhesion molecules; neurexin is expressed presynaptically and selectively binds to neuroligin expressed in the postsynaptic cell

Neurexin interacts with cytoskeletal elements that localize NT-containing vesicles, vesicle release machinery and voltage-gated Ca2+ channels

Neuroligins interact with postsynaptic density proteins that concentrate NT receptors to the postsynaptic side.

Question 22

How are the correct connections made in the developing nervous system? (role of eph/ephrin, DSCAM, and protocadherins)

Answer 22

Multiple signaling mechanisms involved, including…

Eph/Ephrin – there is considerable variability in both the receptor & ligand; they appear to be distributed in a manner indicates a role in labeling synapses specificity

DSCAM – a cell adhesion molecule, so proteins expressed on the pre- and postsynaptic cell are binding with each other (see slide 7)
In Drosophila it has over 38,000 isoforms due to alternative splicing and which isoform is expressed determines which cells synapse with each other. It is actually heterophillic binding that leads to synaptic connectivity; homophillic binding actually leads to repulsion (studies in the Drosophila retina).

Protocadherins – again multiple splice variants lead to a large diversity of isoforms (in the extracellular regions of the proteins)

Protocadherins expressed in the pre- and postsynaptic cells bind to each other (different from cadherins) based on how similar the isoforms are to each other (the more similar the higher affinity for binding)

Not universally expressed at all synaptic sites within a single neuron so may be important for giving each synapse a distinct identity; this would be important for distinguishing on a single neuron which synapses will be GABAergic vs. glutamatergic

Question 23

What is synapse elimination?

Answer 23

refers to the reduction in the number of inputs to a target cell, not a reduction in the overall number of synapses.

Early in development target cells are innervated by multiple inputs and there is a competitive interaction for which synapse “wins” (remains connected) and which synapse “loses”

Question 24

Where are the signals for synapse elimination from?

Answer 24

This competitive interaction involves bot signals generated from both the pre- and postsynaptic cell

In the developing NMJ, blocking either postsynaptic activity (curare) or presynaptic activity (TTX) will result in polyneuronal innervation persisting.

The “losing” axon begins to withdraw presynaptic terminals; this is accompanied by receptor loss on the postsynaptic side (again, an interactive process)

Eventually the losing axon completely withdraws and the remaining axon takes over the abandoned region (ultimately an increase in gross number of synapses)

Question 25

How do curare and TTX relate to synapse elimination?

Answer 25

adding curare and blocking the postsynapse

or adding TTX and blocking te presynapse

= persistant polyneuronal innervation

Question 26

What are neural trophic factors called? What are they?

Answer 26

Once synapses are formed, neurons become dependent on the continued presence of their targets (neuron, muscle, endocrine organ) for their survival

neurotrophins are secreted by their target

This dependence is due to the target cells secreting signaling molecules called neurotrophins (these also contribute to growth and differentiation)

Question 27

Why do we need neurotrophins?

Answer 27

During development in vertebrates, the nervous system makes 2-3X more neurons than are needed.

These extra neurons are eventually culled based on whether or not they have successfully formed connections with the proper targets.

Those that have not, are not exposed to target-released neurotrophins and die as a result of programmed cell death (apoptosis)

Question 28

Hamburger/Levi-Montalcini experiments

Answer 28

In developing chick embryo, there is initially an excess of α-motor neurons produced that eventually disappear.

If the limb bud is ablated on one side, subsequently no motor neurons survive in the ventral horn on the ablated side due to the absence of the target tissue

If an additional limb bud is transplanted onto one side, the number of surviving motor neurons increases because now there is additional target tissue to be innervated

Question 29

three functions of trophic factors

Answer 29

1. Mediate survival of a subset of neuron from a larger pool
2. Mediate the formation & maintenance of the correct number of synaptic connections
3. Support the growth and branching of axons an dendrites to support these connections
   * Again, these trophic factors are made by the target cells and are made in limited enough quantities that there is competition among the input cells for sufficient amounts of these molecules.*

Question 30

Name some examples of neurotrophic factors and explain how cells respond to them (general).

Answer 30

NGF (Nerve Growth Factor) was the first neurotrophin identified.

Others include BDNF (Brain-Derived Neurotrophic Factor), NT-3 (neurotrophin-3) and NT-4/5 (neurotrophins 4 & 5)

Each of these is from a distinct gene that is part of a related family of proteins (trophic factors)

Not all cells in the nervous system respond to the same set of neurotrophins and not all targets produce the same neurotrophins.

This may help to produce some of the specificity for what neurons innervate what targets.

Question 31

How do neurotrophins contact and influence the neuron? What receptor is involved?

Answer 31

Neurotrophin influence is localized to the region of the neuron that they make contact with (see part A).

This can also be seen by virtue of the localized Ca2+ signals that neurotrophins elicit in developing neurons (see B).

Neurotrophin signaling is primarily mediated by Trk (tyrosine kinase) receptors.

Neurotrophins are released in an unprocessed form that undergoes proteolytic cleavage extracellularly; it is this final, processed form of the neurotrophin that actually binds to the receptor

Once bound to the Trk receptor, the ligand-receptor complex in internalized and the now active Trk can mediate the appropriate intracellular signaling cascade either locally or as part of a “signaling endosome” that can be transported back to the nucleus.

change can be profound or localized

Question 32

There are 3 Trk receptors. How does this factor in to neurotrophin signaling?

Answer 32

There 3 distinct Trk receptors with affinities for different types of neurotrophins.

Again, the pattern of what neurotrophins are secreted by the target tissues and what Trk receptors are present on presynaptic neuron can contribute to the specificity for how neural circuits are formed.

Trk receptors interact with different downstream signaling pathways that have a variety of functional roles.

Question 33

Things to know about p75.

Answer 33

The p75 receptor is another neurotrophin reeptor (part of the TNF family).

It is a low affinity receptor for neurotrophins (not as sensitive as Trk)

It lacks the specificity of Trks and seems to respond to all neurotrophins

It actually responds to the unproceassed forms of the neurotrophin proteins.

As with Trk, p75 receptors activate a variety of intracellular pathways associated with different functions.