Lecture 33 mcb60

80%

Around the time that their axons are invading their targets

• Around 1920s
• Looked at the effect of removing or adding a limb (TARGET) on motor neuron survival in the spinal cord

• Normal survival: 50%
• Removing: REDUCES (to 10% in experiment)
• Adding: INCREASES (to 75% in experiment)
• Suggests: that the target produces some factor in limiting quantities that promotes motor neuron survival

• Programmed cell death
• Motor neuron death

• That target cells release a factor (present in limiting quantities) that promotes cell survival
• Neurons reaching the target compete for these factors in order to survive

Nerve growth factor

• Found that mouse sarcoma tumor release a factor that promotes survival of DRG and sympathetic neurons in culture
• NGF: they isolated this factor and called it nerve growth factor
• They found that neurons die in the absence of trophic factor (in this case NGF)

NGF

Trophic factors that promote neuronal survival/prevent cell death

• Add factor to culture: neurons live
• Remove or block its function: neurons die

Neurons DIE

Neuron SURVIVE

Neurons DIE

• Neurons SURVIVE
• NOTE: cell death dependent on protein synthesis

• • Neurotrophins
• • TGF-Beta family
• • Interleukin-6 related cytokines (LIF, CNTF)
• • FGFs

• NGF: TrkA receptor
• BDNF: TrkB receptor
• NT3: TrkC receptor
• NT4/5: TrkB receptor
• NOTE: these all also bind p75 receptors, and they can bind the other receptors but with lower affinities

YES

NGF on skin

NT3 on muscle

GDNF, FGF, HGF on muscle spindle

NT3, GDNF, NGF on viscera

Neurons DIE

Neurons DIE

Neurons SURVIVE

Apoptotic vs necrotic cell death

• Apoptotic: INTRINSIC factors, suicide
• Necrotic: EXTERNAL factors, injury or murder

• Intrinsic
• • Genetically programmed
• • Cell shrinkage
• • Chromatin condensation
• • Cellular fragmentation
• • PHAGOCYTOSIS (eaten from inside out)
• • Programmed cell death

• External factors
• • Trauma/injury
• • Messy
• • Lytic
• • Inflammatory response

Neurotrophic factors

No: Pathways and molecules of apoptosis have been conserved through evolution

• Ced-3: in roundworm C elegans
• Caspase: in humans
• NOTE: these are PROTEASES (effector)

• Ced-4: in roundworm c elegans
• Apaf-1: in humans
• NOTE: these ACTIVATE Ced-3 and Caspase (which lead to cell death) so they’re PRO-apoptotic

• Ced-9: in roundworm c elegans
• Bcl-2: in humans
• NOTE: these INHIBIT the ACTIVATORS so they are ANTI –apoptotic

• Egl-1: in roundworms c elegans
• BAD: in humans
• NOTE: These INHIBIT the INHIBITORS so they are considered PRO-apoptotic

PROMOTE . . . INHIBITING

• A sensory-motor structure that recognizes and responds to guidance cues
• The expanding tip of the axon: it’s an enlargement at the end of growing axons

• It is the expanding tip that is a sensory-motor structure that can recognize and respond to guidance cues
• by promoting growth towards or away from those cues or signals

It is full of MICROTUBULES at its core and ACTIN at the leading edges

• Lamellipodia
• Filopodia

• Lamellipodium: broader, foot-like or sheet-like projections made of branched actin that represent the ‘’leading edge’’ of the cell
• Function: help treadmill the axon forward

• Filopodia: finger-like structures made mostly of actin that extend out of the growth cone and beyond the lamellipodia
• Function: responsible for sensory ability of the growth cone

• Vesicel fusion and Actin polymerization pushes the filopodia forward
• This will drag the lamellipodia and the microtubules from the center core to advance, creating new regions of the axon

• ATTRACTIVE cues: will reduce F-actin assembly.
• This will promote filopodia growth.
• REPULSIVE cues: will reduce filopodia growth

ACTIN

MICROTUBULES

ACTIN like filaments

• Microtubules
• Mitochondria
• Vesicles

ACTIN

• A drug that binds to actin filaments and prevents their polymerization
• Function: add it locally to the growth cone, quickly inhibit movement

• Contact mediated
• Long range

Using DIRECT contact, the axon can adhere to parts of extracellular matrix, cell surfaces, other axons (fasciculation), or be repulsed by contact (contact INHIBITION)

• Axons can use long range signals, like gradients of signaling proteins.
• This will lead to chemo-ATTRACTION or chemo-REPULSION

• 1) Laminins
• 2) Integrins

• Laminins: Major components of basal laminae
• Extracellular matrix: is where theyre found
• Accounts for: much of the axon outgrowth promoting ability of the extracellular matrix

• Alpha, beta, and gamma subunits
• 5alpha, 4beta, 3gamma genes

• Integrins: Another contact mediated protein that guides axons
• Growth cone: is where they’re expressed/found
• Laminins: is what they interact with

• Alpha and beta subunits
• 16alpha and 8beta genes
• Different heterodimers interact with different heterodimers of the laminins

• Cell surface attraction: is what theyre responsible for
• Examples: Cadherins and Immunoglobulins

Expressed in both the GROWTH CONE and the cells that are mediating the direct contact attraction

Means the Cadherin or Immunoglobulin expressed by the GROWHT CONDE is attached to the same Cadherin or Immunoglobulin in the CELL SURFACE of the attracting cell

It’s a topographic map of the visual field from the retina to the tectum (chick, frog, fish) or to the superior colliculus (mammals)

TECTUM

The RETINA

• There is a point-to-point map from the retina, it’s just INVERTED over the A/P and D/V axes.
• Both the retinal and Tectum are rightside up, dorsal on top and ventral on bottom, but the image crosses in the middle so that the image is inverted or upside down

There has to be signaling coming from the specific regions of the tectum or SC to tell the neurons (projecting from the retina) where to go

• 1) to leave the retina at the optic nerve head (ONH)
• 2) To cross (or not) at the optic chiasm (and where)
• 3) A/P and D/V positions

• Pre-target formation: the axons (dendrites?) just shoot out from retina to tectum
• Branch formation: branches form and they find where they need to go
• Final refinement: branches have found their location and everything is nice and neat

Retinal ganglion cell

• In their TARGET ZONE (TZ)
• Some take direct path, some overshoot and come back

• Roger Sperry: da man of 1963
• Hypothesis: states that there must be a unique molecular address in the tectum or SC
• Also that each RGC that projects to the tectum of SC has a unique set of receptors for the tags expressed by the tectum/SC

RGCs still make connections according to their original (intrinsic) positions

• 1) Molecular address/tag: Each position in the optic tectum has a unique MOLECULAR ADDRESS or MOLECULAR TAG
• 2) Identity: Each RGC has a unique set of receptors for these tags (i.e. an identity) resulting in a position-dependent response dependent on differential affinities or differential intracellular responses
• 3) A/P, D/V: unlikely to be unique molecules for each position (would be insufficient info in the genome to wire up entire brain this way), rather, info is probably encoded in orthogonal gradients

Positional identities of axons and targets are matched up to establish the point-to-point topographic map

• If you were to rotate frog eyes 180 degrees, the projections of retinal will be maintained
• Cells that were originally ventral projecting dorsal will still project dorsal even when switched to a dorsal position
• Conclusion: where the cells is MADE determines where it will project to, not the position from where it grows
• I hope this makes sense to you :/

• Scientist who developed the Stripe Assay to demonstrate sensitivity of temporal retinal axons to a repellent activity in posterior tectal membranes
• To test the affinity of the different projecting retinal ganglion cells in the retina

• He wanted to design a unique way to culture developing RGCs
• • He would make ALTERNATING STRIPES of cell homogenates from either the posterior or anterior portion of the tectum
• • These stripes would contain the FACTORS expressed in the posterior or anterior parts of the tectum
• • He would then GROW different parts of the retinal tissue, either TEMPORAL or NASAL

• Temporal: is considered posterior and it will project anteriorly in the tectum
• Nasal: is considered anterior and it will project posteriorly in the tectum
• TP?
• NA!

Repulsive Axon Guidance Signal

• He found that if we exposed axons from posterior/temporal region of the retina, it would preferentially grow in the region of the plate that had anterior portion of the tectum
• The anterior/nasal region didn’t have a preference

That there was a repulsive cue expressed by cells in a specific region in the tectum (e.g. posterior) that only affected cells expressed by that region of the retina (e.g. posterior/temporal)

• GPI: Glycosyl Phosphatidyl Inositol
• PI-PLC: Phospho Inositide Phospho Lipase C
• RAG: Repulsive Axon Guidance

• By administering PI-PLC which meant that the signal, or protein, they were looking for had a GPI anchor
• Eph Receptors and Ephrins: The protein they purified, RAG, ended up being part of a larger family of proteins called Eph receptors, and their targets, Ephrins

This is the family that RAG is a part of

Ephrin A5 protein

To be expressed in a complimentary manner

• LESS
• So if the gradient of the expression of ephrinB goes from HIGH dorsal to LOW ventral, the cells in the dorsal portion will be very sensitive to EphB receptors bc they express so much of the ligand
• So if those cells then project to the tectum, they will grow only into low gradient (bc they are so sensitive and will be inhibited from growing up the gradient, bc even small amts will inhibit growth)

Chemoatractants or chemorepulsants

Collapse and attempt to grow DOWN the gradient of the chemorepulsant

A family of chemorepulsants

• Collapsin-1
• Func: it induced the collapse of the growth cone, hence the name
• NOTE: they don’t induce growth cone collapse on ALL neurons, so they act more as signals for growth

Temporal axon growth cones

Repellent activity

In the retinal ganglion cells