L3: Layer-specific Targeting and Topographic Maps

Why are topographic maps needed

• so the brain can integrate a multitude of stimuli from the environment

• formed in the sensory and motor areas

Discovery example of somatotopy 1

John Langley: Reflex-mediating neurons of the superior Cervical ganglion Topographically ordered:

• If activate neurons with axons projecting ot of the top thoracic nerve root→ pupil reflexb

• If activate neurons with axons exiting the fourth thoracic nerve root→ Dilation and constriction of the blood vessels of the ear

THEREFORE: an order→ topogrphical map

What did Langley also find

• Cutting of the preganglionic nerve above T1→ abolishes all reflexes

• Upon axonal regneration→ reflexes are re-established eccuratley

What this suggests→ the region itself has some kind of order→ somehow marked and find eachother again to be regenerated

• prior order is restablished

Discovery example of topography 1

Wilder PEnfield

• Used electrodes to systematically probe different areas of the cortex of patients who were consicous during the operation

result:

• Found motor and sensory cortical areas

• BOTH organised to for somatotopic maps

Neural maps what are they

• internal representations of the body and/or the outside world

• Maps can be discrete or continuous

Neural maps where can they be found

• found throughout the nervous system

• for sensory→ auditory, visual, olfactor maps etc

• for motor systems

Why are they useful

• constitute a fundamental organising principle

• strategy for organising and presenting synaptic information

• facilitate complex neuronal wiring of populations of neurons by providing:

  • order to the spatial relationships

  • and/or

  • qualitative relationships
    

Types of maps

1. Spatial

   • preservation of the nearest neighbour

   • anatomical representation within the brain

   • e.g Homunculus→ nearest relastionship of the body represented

2. Quality map

   • e.g types of smell or taste

   • widley distributed over the receptors (epithelium)

   • but the terminals cluster together in a functional sense→ depending on quality

Example of targeting axons to discrete regions→ example

• The Motor system

The vertebrate neuromuscular system→ overview of how cell types specify

• axon pathfinding seen as a sequence of simple choices

• progressively define axon tragetories and target areas

• restrict the developmental potential each time and become specified

What is this decision process in vertebrate motorneurons

Each dscision is binary

1. Start off generic

2. choose visceral or somatic

3. somatic→ LMC (limb) or MMC

4. LMC→ medial or lateral

5. contiues

How is the decision between motoneurons that innervate dorsal limb and those for ventral limb made

• Dorsal limb musculature→ located in the lateral part of the LMC)

• ventral limbs muscles→ located in the medial part of the LMC

Distinction between these is generated by birth order:

1. mLCM are born first→ Express the

   • Lim-Homeodomain (contains both LIM and Homeobox motifs) Transcription factor Islet1

   • and synthetic enzyme Retinaldehyde Dehydrogenase-2 (RALDH2)

2. mLCM make RA (retinoic acid)

3. so the later born lLMC neurons are exposed to RA

4. causes lLMC to express different transciption factor→ Lim1

5. causes different surface proteins

   • So cluster together in different lumps

What maintains the identity of these two seprate sets of cells

→ Cross-repressive interactions between islet-1 and Lim-1 maintain a stable distinction between these two sets of cells

To make Motoneurons innervating axial muscles (MMC in stead of the LMC)

• Different Lim-Homeobox transciption factor→ Lim-3 (aka Lhx-3)

Overall what does this image show how cell identity is made

→ Expression of different combinations (codes) of lim-homeodomain transcription factors

therefore: it is not a single TF that is involved→ kind of a combination

• this is useful because it allows great diversity to be made with a small molecular code

  • (can see in the image that there is repeated use for the same TFs→ but just different codes)

• Rather than having a TF expressed for every single different neuron

• These codes are made due to birth order

What do these Lim-homeobox TFs also specify for on top of motor neuron identity and how found

• Specify distinct axonal trajectors

• into distinct target regions in the periphery

How found

• Knockout/knockdown and mis-expression of these the lim-homeobox TFs in mice and chick

This axonal specification may be conserved→ evidence

Compared to the vertebrates→ Drosophila embryo combinatorial code

• Also for Lim-Homeobox transciption factors:

  • Islet and Lim3

• Motor axon trjectories are also specified

  • by POU domain factor→ Drifter

How do these Lim-Homeodomain codes actually cause their effect on identity?

• Lim-Homeodomain codes regulate Eph-receptor expression

  • Islet-1→Eph-B1

  • Lim-1→Eph-A4

What do these receptors do

• Regulate the simple binary choice of whether axons innervate the dorsal or ventral limb

• How→ be responding to different gradients of guidance cues found in the limb? (ephrin-A and ephrin B2)

  • mLMC→Islet-1→Eph-B1→ ephrin-B2 ATTRACTION→ ventral limb

  • lLMC→Lim-1→Eph-A4→ EphrinA INTERACTIONS→ dorsal limb

How do these guidance cues work for vertebrate limb

Guidance cues are in complementary zones in the limb:

• Ephrin A→ in the ventral

• Ephrin B2→ Dorsal

1. lLMC→ Eph-A4→ repelled by Ephrin A→ goes to the dorsal

2. mLMC→ Eph-B1 receptor→ interactis with ephrin B2→ goes to ventral

Complemntary zones of Eph-ephrin expression

How is the high fidelity of the neuromuscular connectivity achieved?

• employing mutliple of these targeting mechanisms simultaneously

Invertebrate Neuromuscular system→ Drosophila structure overview

• 30 motor neurons to 30 muscules

• 1:1 innervation

• different peripheral nerves deliver sets of motor axons to different target regions

How is this mapping set up?

1. sets of muscles and their innervating motor axons express matching homophilic Cell Adhesion Molecules (CAMs)

   • e.g Fascicilin-3 and Connectin

2. Also, some muscles also express secreted guidance cues

   • e.g netrins or semaporins

What the combination of these things do

1. guidance cues modulate the CAM-mediated attraction

2. And thus→modulate overall balance of attractive and Repulsive forces

   • growth cone picks up on a there differences and makes a decision

3. overall: determines the choice of Target muscle

Investigating how these choices are made

• Altering the combinatorial code

Procedure:

• Change the concentrations of NET and SEMA

• see where the neurons project to

• can see how the balance of forces due to NET and SEMA change the course of the pathway

Overall summary of the organisation of the neuromuscular system

1. growth cone guidance of motorneurons mirror the process of cell fate decisions→ hierachical sequence of binary decisions → specificity

2. Different TPs→ different guidance cue receptors→ different response to existing guidance cues

3. Integrate musliple guidance cues (secreted and membrane bound)

   • balance of repulsive and attractive

   • → overall confer preferences and specificity to the type and area of the motor neuron

Note: Channelling growth cones into distinct nerves and thus towards specific target regions limits what

→ Partner choice

• If you channel a different number of motorneurons- >you will get different choices and outsomes

• chanelling is about restriction of choices

Therefore just remember that: although the TFs, receptors and their responses to guidance cues has an impact on the identity, you also have the factor that with every choice made, another neuron’s choice will also be effected. The number of neurons available will mean that the combinatorial codes will become different outcomes?

• i.e all affect eachother?

Example of continuous maps

• retinotopic projections

• unlike motor system→ not 1:1 mapping

How are continuous neural maps formed?→ e.g the Retinotopic map

• retinal axons→ optical tectum (project through retinal ganglion cells)

  • relative position on retina is conserved on the tectum:

  • Temporal retina→ Anterior tectum

  • Nasal Retina→ Posterior tectum

: retinal ganglion cells project ro specific regions of the tectum so that neighbouring cells in the retina will form connection next to each other in the tectum, with neighbouring tectal partner neurons

• .I.e map of visual space is preserved in the brain

Before the mid 1900, what was thought as to how thse connections were set up?

• outcome of trial and error

• with functional validation

  • sorted neural connections according to function

Roger Sperry experiments to challenge this view

Procedure 1:

• rotated eyes of a frog by 180 degrees (took eye out and back in)

• left optic nerve intact

• test behaviour of animal

Result:

• frog saw world upside down

Procedure 2: (done to test the re-generative effect)

• rotated frog eye 180 degrees

• cut optic nerve

Result:

• saw world upside down

• irreversible

Two conclusions from these experiments:

1. Precise retinotectal connectivity was not directed by experience→ must be an anatomical feature

2. retinal axons regenerated after the optic nerve had been cut→ grew back into the tectum and there re-establised synaptic connections at about the same location that they had previously occupied

   • According to the original anatomical coordinates in the eye

From these conclusions→ Sperry’s chemoaffinity hypothesis

• every retinal axons has a particular chemical affinity for a particular location (and thus postsynaptic target) in the tectum

  • this would explain why there is no functional effect and how it regenerates to the same place as before

    • because it is under the same influence of chemical affinity stuff

What did Sperry postulate as to how this chemoaffinity hypothesis worked:

• a multitude of retinal axons could be mapped onto the optic tectum by two or more perpendicular cytochemical gradients

• i.e there are two orthogonal gradients

  • one in the retina

  • And one in the tectum

• Match up toegther to conserve the retinotopic map as the neurons target onto the tectum from the retina

But what are the mechanisms of this hypothesis?→ next question to ask

• how is it that both presynaptic retinal and posynaptic tectal neurons establish their positional identities?

Investigating how these maps are made→ could temporal differences in differentiation and targeting establish the map

Procedure: See when/where the the RGC in the dorsal and ventral arrive in

• WT

• Retina with dorsal side replaced with another ventral (double ventral)

Results

• Wild-type→ Dorsal Root Ganglion cells arrive first

• double ventral→ ventral RGC→ select their normal target area

Conclusion:

• it is not about the timing of when the RGCs

• there is some kind of patterning

• it is not just about which part if filled up first

  • (other wise you should get the WT phenotpye in the double ventral one)

The positional information that confers a retinotopic identity seems to be established when?

• during the development of retina and tectum

1. How Temporo-nasal polarity is set up in the retina (in chicks)

1. Expression of homeobox transciption factors in non-overlapping domains

   • BF-1 and BF-2

2. These confer some positional information to retinal ganglion cells in the Antero-posterior axis

1. How Antero-posterior polarity is set up in the retina (in chicks)

• Gradient expression of Engrailed (En) (works as a transciption factor)

  • high En→ posterior tectum

  • low En→ anterior tectum

How was TF engrailed found?

• testing RNAs→ cDNA

• Find a TF and Apply

• see where the axons go (A or P)

• found engrailed

Testing the effect of high and low levels of Engrailed expression

Procedure:

• misexpress engrailed using viruses

• misexpress in different patches of cells

Result:

• High levels of ectopic Engrained expression =

  1. Attract branches of Nasal axons

  2. Repulse temporal axons

• Therefore→ different ganglion cell axons from different places in the retina have a different response to high levels of engrained

• engrailed must be regulating guidance molecules for retinotopic mapping

How were guidance molecules used for retinotopic mapping (that engrailed might be regulating) investigated

Bonhoeffer

→ Microscopic carpets→ striped carpet assay

• alternating stripes of membrane derived from either anterior or posterior parts of the tectum

How are these striped carpet assays made

• put nasal or temporal axons in stripes

• onto anteror and posterior regions of the tectum

• See where they want to go

Results from the striped carpet assay

1. Temporal retinal axons→ prefer Anterior temporal membranes

2. Nasal retinal axons→ no preference

Investigating further→ finding the biochemical components that restrict the temporal retinal axons to the anterior only

Procedure:

• heat treat specific membranes

• or→ incubate with an enzyme (PI-PLC)

  • destroys phosphotidyl-inositol (PI)linked membrane molecules

Result:

• If disrupt posterior tectal membranes—> Temporal axons now also go to the posterior

  • loss of preference for the anterior

conclusion:

• There is repulsion from the posterior tectal on the temporal axons

what was this repulsive function found to be?

• mediated by a member of the family of Ephrin (Eph) ligands

• these set up biochemical gradients

What kind of molecules Eph ligands

1. A-type→ GPI-linked molecules

2. B-type→ Transmembrane molecules

Receptors for these ligands

• Have a large family of receptor tyrosine kinases

• Correspond to A or B types

Therefore

• All A-type Ephrin ligands→ activate all A-tpye Eph receptors

• All B-type Ephrin ligands→ activate any B-type Eph Receptor

i.e specific ligands go to specific recetprso (similar to what is seen in motor neuron?)

If all e.g type A can interact with type B→ how do you get a wide scope of signalling?

• Ligand-receptor pairing have differential affinities for one another

What are the biochemical gradients of ephrin in the tectum

• A2 and A5

• High levels → Posterior

• Low levels → Anterior

i.e must be the repulsive force for getting temporal axons to the anterior

Therefore what is the retinal axon receptor pairing to the tectum biochemical gradient (i.e how is temporal retinal ganglion to the posterior)

• Temporal retinal ganglion cells→ Express high levels of EphA3 receptor

• receptor for the high levels of the A2 and A5 in the posterior tectum

• Causes→ repulsion of the posterior

Further evidence that ephrin A2 repels the temporal axons:

1. when ephrin-A2 is virally over-expressed at high levels throughout the tectum

   • → temporal retinal axons fail to make terminal projections into the tectum

2. When under-expressed (ephrin-A5 knockout)

   • → ectopic arborisations of termporal retinal axons in the posterior part of the tectum

   • i.e no longer being repelled from the posterior

Overall this shows us that

• repulsion is key to topographic map formation

but this only explains the temporal axon pathway

How are these gradients of ephrin A2 and A5 expressed?

• Due to the expression profile of En (Engrailed)

• Indeed this has been proven

• therefore shows how the TF is setting up these gradients

But questions that have not been answered

1. How are nasal RGC axons patterned?

   • computational models predict a requirment for at least two opposing forces for topographic mapping

2. What actually provides the opposing force?

   • competition for space or trophic factors?

   • Attractive cues?

3. Why do retinotopic maps still form in the absence of ephrins??

4. Repulsion does not cause precision→ how does it get so prescise?

1. How are the retinotectal projections of the nasal axons patterned?

• EphA-ephrinA interactions actullay have different effecst at different concentrations

  • higher concentration→ repulsion (for temporal)

  • lower concenctration→ prmote retinal axon growth

therefore shows: dual concnetation dependent response

• guidance cues can mediate both forward and reverse signalling

• therefore: this signalling system can provide the requirred two opposing gradients through

  • differing receptor responces from differeing concentrations

  • overall→ can get temporal and nasal mapping from the same gradients!

3. Why do retinotopic maps still form in the absence of ephrins??

• other signalling molecules are present in the developing visual system

Example→ TF Engrailed itself

• also acts as a guidance cue for retinal axons:

  1. Taken up by growth cones of Xenopul reintal ganglion cells

  2. initiates translation of new proteins and chemotropic response:

     • Temporal→ repulsion

     • Nasal→ Attraction

i.e→ we again get two forces from the same one gradient

How it was found out how Engrailed worked

Procedure:

1. Control→ En-2 internalised and acts as a guidance cue (rather tahn a TF

2. Mutating the internatision ‘penetratin’ domain→ abolish engralied protein internalisation→ abolishes effect on growth cone guidance

Conclusion

• Engrailed is internalised→ so does act as a guidance cue and not as a TF in this instance

Therefore the presence of engrained 2 explains

• how there is still mapping in the absence of ephrins

• its attractive mechanisms may aid the precision needed

  • (which sn’t really provided by just repulsive mechanisms)

Patterning of retinaltectal projections in the D-V axis relies on

Attractive rather than repulsive ephrin/Eph interactions

Problem→ during development, the size and shape of the tectum changes

• this means that even though there is retinotopy estblished at the start of development

• this could be disrupted at the tectum itself grows

but→ it is not!

What does this suggested about the retino-topic connections made

• have to be flexible

• so they can be adjusted continually

Experimental manipulations demonstrate the capacity for adjustments of connections

Procedure: see where axons grow in tectum manipulation

• Control

• Half tectum

• Half tectum and then regulated with cues

Result:

• Half tectum→ temporal axons want to not be in the posterior part (removes somatotopy)

• regulated→ get somatotopy

Experimental manipulations demonstrate the capacity for adjustments of connections→ retinal

Procedure: see where axons grow in retinal manipulation

• Control

• Nasal removed

• Nasal removed and then regulated with cues

Results:

• nasal removed→ none to the posterior

• Regulated→ regains the somatotopy

Two experiments show two pahses of this flexibilty

1. Targeted growth, directed by guidance cues

2. Process of adjustment, partly through compeitive interactions between axons

What are the relative contributions of these two forces (cues vs competition)→ how investigated

Procedure: studying single retinal ganglion cells develping in isolation

• transplantation of single, labelled WT cells into zebrasih ‘lakritz’ mutants

• these fail to form retinal ganglion cells

Results and conclusions

1. normal positional of distal (posterior) terminals in tectum according to position in the retina

   • → Eph expression levels

2. Abnormal expression proximally (Into anterior tectum)

   • → suggests: interaxonal competition normally refines arbor territory

Summary of this

1. retina and its target tissue are polarised

   • due to expression of patterns of

     • TFs and Guidance cues

2. What generates the retinotopic map in the early embryo

   • gradients of guidance cues and receptors

   • in retina and tectum/superior colliculus

     • (Eph/ephrin; Wnts;Engrailed)

3. What refines the coarse maps initially formed

   • neural activity and other compeitive mechanisms