Axon Guidance and Synaptogenesis

Module 404

Sean Sweeney

Aims and outcomes:

To understand how neurons develop from an undifferentiated state to a complex morphology.

To understand the mechanisms that neurons use to grow in appropriate directions to find the correct partners and generate the ‘wiring diagram’ that constitutes the functioning brain.

To be aware that different molecules expressed during the process of neuronal differentiation generate neuronal diversity AND molecular specificity to organisethe ‘wiring diagram’.

Undifferentiated neuronalcells grow to become morphologically distinctand functioning nerves….

….making appropriate connections with correct synaptic partners in distinct areas of the brain to form circuits.

How do growing nerves generate the final wiring diagram?

Number of neurons in the human brain:20,000,000,000 to 50,000,000,000

Number of synapses: 1014

Number of synapses per neuron: 2000 to 5000

How does a genetically programmed system organise thiscomplexity?

Neural induction, migration, determination and differentiation(lectures in module 301)

Axon outgrowth (301)

Axon guidance (301)

Target selection

Synaptogenesis (formation and function)

Synapse refinement (addition and subtraction)

Behavioural development

Neuronal ‘stereotypy’ identified by Ramon y Cajal andothers (ca. 1890-1910)

Coghill and others (1929) ‘individuation vs integration in the development of behaviour’ : Neurons, by their activityand ‘learning’, select the correct connections duringdevelopment. ‘primitive thrashings of developing organisms’.

The Chemoaffinity Hypothesis:

Sperry, R.W. (1943) J. Expl. Zool. 92: 263-279 ‘Effect of 180degree rotation of the retinal field on visuomotor coordination

The Chemoaffinity Hypothesis:

Severing the optic nerve, rotating the eye 180 degreesand allowing the nerve to regenerate results in visuomotorimpairments in the frog (Sperry)

The Chemoaffinity Hypothesis of Sperry:

1. Axons have differential (biochemical) markers

2. Target cells have corresponding markers

3. Markers are the product of cellular differentiation

4. Axonal growth is actively directed by markers to establish specific connections

It follows that: The code for axon guidance is ‘hard-wired’ (GENETIC!)

There is an order to the code

The Chemoaffinity Hypothesis Cont:

Uncrossing of opticnerve fibres followedby nerve regenerationleads tovisuomotor defectsin frogs

(importance of a ‘midline’ choicepointthe brain is bilaterallysymmetric)

Growth cones are active and dynamic projections rich inmicrotubules and actin filaments

“The cone of growth is endowed with amoeboid movements. It could be compared with a living battering ram, soft and flexible, which advances, pushing aside mechanically the obstacles which it finds in its way, until it reaches the area of its peripheral distribution.”Santiago Ramon Y Cajal

Guidance Cues:

Target derived (positive and negative cues)

Local vs long range (diffusable vs cell attached in theextra-cellular matrix)

Time dependent

Actin cytoskeletondynamics can be regulated by small monomericG-proteins

Rho - induces stressfibres

Cdc42 - inducesfilopodia

Rac - induceslamellipodia

Fibroblasts transfectedwith a small G-proteinand stained for actin(G-protein is engineered so that it cannot hydrolyseGTP and is thereforeconstitutively active)

wild type(untransfected)

Cdc42 Rac Rho

Summary

Axons can use manycues and combinations ofcues to guide them to their correct location.

These cues are interpretedby the growth cone as theperceived cues act to regulate the actin cytoskeleton and determine the directionof the growing axon

Neural induction, migration, determination and differentiation(lectures in module 301)

Axon outgrowth (301)

Axon guidance (301)

Target selection

Synaptogenesis (formation and function)

Synapse refinement (addition and subtraction)

Behavioural development

Guideposts/choicepoints

Ti1 pioneer axons ingrasshopper embryo(Bentley and Caudy 1983)

Dictinct identifiable cells act as local routemarkers to give direction to growing pioneer axons

Contact mediated attraction

Growth cones adhere to substratecell upon detection of a positivecue (a cell surface molecule)

Mediated by:CAMs (IgG superfamily proteins)cadherinsephrins/Eph receptorsintegrins

Contact mediated repulsion

Growth cones retreat froma cell upon detection of a negative cue (a cell surface molecule)Mediated by:collapsins/semaphorins

growth

growth

The collapsins/semaphorins

Chemoattraction

Long distance cueSecretedMediated by:Nerve Growth FactorNetrin/DCC/unc5 interaction

Gradient of secreted cue

The Netrins/DCC/unc5

Chemorepulsion

Long distance cueSecretedMediated by:slit/roundabout interactionsemaphorins/collapsins

Gradient of secreted cue

Slit/roundabouts

The Drosophila embryonic ventral nerve cord

Drosophila embryo side view

anterior posterior

ventral view

dorsal

ventral

Fasciculation: pioneers vs followers

Followers can fasciculateand de-fasciculate and usecomplex combinations of cuesto do so

Trophic support, a mechanism for regulating numbers and direction of growth cones

growth

Gradient of Nerve Growth Factor

Competing growth cones

Target cellSecreting NGF

Trophic support, a mechanism for regulating numbers and direction of growth cones

growth

Gradient of Nerve Growth Factor

Competing growth cones

Growing nerves that receive insufficient NGF die by a processof programmed cell death (aka apoptosis)

Target cellSecreting NGF

The Nerve Growth Factors/Trk receptors

Neural induction, migration, determination and differentiation(lectures in module 301)

Axon outgrowth (301)

Axon guidance (301)

Target selection

Synaptogenesis (formation and function)

Synapse refinement (addition and subtraction)

Behavioural development

Dendritogenesis: 1st step, determine polarity:

One neurite predominatesand becomes the axon,others become the dendrites.Thereafter, guidance cues may be similar to thoseguiding axons, growth occursin similar timewindowDendrites may also utilise‘tiling’.

The Drosophilalarval body wallis innervatedby sensory dendritesof many differentclasses(Grueber et al., 2002Development, 129;2867-78)

Sensory dendritesoccupy territories that Exclude dendrites of the same sensory class. Ablation identifies a mutual inhibitionthat ensures efficient ‘tiling’of the body wall surface.Also occurs in zebrafish

‘Heteroneural Tiling’

Target selection and synaptogenesis.

Dscam: determining adhesivity and diversity

In Dscam nulls, all terminal arbours fail to develop. In mutants lacking various splice forms, many terminal arbours are lacking.

Dscam generates diversity and specificity of connections (Bharadwaj and Kolodkin (2006) Cell 125, 421-424)

Each neuron expresses a small and distinct subset of alternatively spliced DSCAM isoforms required for the recognition of ‘like’targets.

Grueber paper DSCAM

Dendrite ‘self’-avoidance contributes toefficient tiling: isoneuronal recognition

DSCAM mediates isoneuronal recognition by an inhibitory mechanismregulated by the C-terminal of the protein (see Zinn, K. (2007) Cell 129, 455-456

Neural induction, migration, determination and differentiation(lectures in module 301)

Axon outgrowth (301)

Axon guidance (301)

Target selection

Synaptogenesis (formation and function)

Synapse refinement (addition and subtraction)

Behavioural development

Synaptogenesis: what are the cues that induce a synapseto form from a growth cone?

Many of the molecules regulating guidance are alsoinvolved in synaptogenesis: are these cues inductive?

Partner recognition (cessation in growth)?: adhesion moleculessidekicks, flamingo, DSCAM, SYG1, SYG2Shen (2004) Molecular mechanisms of target specificityduring synapse formation. Curr Opin Neurobiol 14, 83-8

Prior to synaptogenesis: transient rise in calcium

Morphological transition from growth cone to synaptic boutonImportance of transport ‘packets’e.g. PTV packets (Piccolo-Bassoon transport vesicle)

immaculate connections (imac): Pack-Chung et al (2007)Nat. Neurosci. 10, 980-989

Signals for synaptogenesis? Agrin?

The mammalianneuromuscular synapse

Acetylcholine receptorsare diffusely distributedacross the muscle fibreuntil the arrival of a neuron

Acetylcholine receptors cluster in response to the arrivalof a neuron: does the neuron promote synapse maturation

Purification of ‘Agrin’, a proteoglycan normally secreted by the neuron, suggested Agrin induced synapsematuration (Sanes et al., (1978) J.Cell Biol 78:176-198)

Agrin deficient neurons fail to Induce neuromuscular synapsematuration

1. Agrin recruits AchRs2. Agrin induces transcription

Of AchRs from ‘synaptic nuclei’3. Transcription of AchRs from

extra-synaptic nuclei is downregulated

4. Rearrangement of muscle cytoskeleton

5. Retrograde signal from the muscle to the nerve to stabilise the synapse

Neural induction, migration, determination and differentiation(lectures in module 301)

Axon outgrowth (301)

Axon guidance (301)

Target selection

Synaptogenesis (formation and function)

Synapse refinement (addition and subtraction)

Behavioural development

Zito et al, 1999

Marking synapses:Live synapse eliminationWalsh and Lichtman(2003) Neuron 37:67-73

Live synapse growth:Zito et al., (1999) Neuron22: 719-729

Aberle et al., (2002) Neuron 33, 545-558

Marques et al.,(2002) Neuron 33, 529-543

witA12/witB11 wt

Synaptic growth is regulated by a TGF-ß type-II receptor wishful thinking (wit)

Neural induction, migration, determination and differentiation(lectures in module 301)

Axon outgrowth (301)

Axon guidance (301)

Target selection

Synaptogenesis (formation and function)

Synapse refinement (addition and subtraction)

Behavioural development:

Bate, M. (1999) Current Opinion in Neurobiology 9:670-5Bate, M. (1998) International Journal of Developmental

Biology 42: 507-9

Reading Material:

Purves et al, 3rd Edition, Chapter 22.Sanes, Reh and Harris., Development of the Nervous System.2nd edition. Academic Press 2006

Bentley and Caudy (1983) Nature 304:62-65Sanes et al., (1978) J. Cell Biol 78:176-198Tessier-Lavigne and Goodman (2001) Science 274: 1123Sanes and Lichtman (2001) Nature Reviews Neuroscience2:791-805Sanes and Lichtman (1999) Annual Reviews in Neuroscience22:389-442Jan and Jan (2001) Genes and Development., 15; 2627-2641