Axon GuidanceHow does an axon find the right target?Regenerating retinal ganglion neurons project to their appropriate positionEphrins are guidance cues (ligands) in the optic tectumEphrin receptors (receptors) are in the retina axons1Axon GuidanceHow does an axon find the right target?• 100,000,000,000 neurons in the brain• 1000 synapses per neuron• 100,000,000,000,000 connections in the brain1How does an axon move?How does an axon know where to go?2The Growth Cone is the expanding tip of the axon 32Properties of Growth Cones- A growth cone is an enlargement at the end of a growing axon. - Contains several finger-like projections that are called filopodiaand sheet-like projections called lamellipodia.- Filopodia and lamellipodia contain actin-filaments.- The growth cone core or central domain contains microtubules, mitochondria and vesicles. 4Axons require actin at the growth cone to extend1) cytochalasin B is a drug that binds to actin filaments and prevents their polymerization.2) Add cytocholasin B locally to the growth cone,quickly inhibit movement5Movement of the Growth Cone63Movement of the Growth Cone1. Actin polymerizes at filopodia tipand depolymerizes at base(provides directionality)2. Microtubules extendfrom the growth cone base(central core)7Retinal ganglia cells synapse in the optic tectum (LGN)1. Most axons stop at the right target2. Some overshoot, turn around and come backModel: There is a signpost that the axon recognizesHow does a growth cone know where to go?8Roger Sperry (1940s-1950s) - formation of neural pathways in the brain is very preciseThe pathways that developing axons take are very accurateRamon y Cajal (1890s)- growth cones move in an ordered and directed manner (Cajal won the Nobel Prize in 1906 for this work). Ross Harrison (1930s)- first observed growth cone movement of neurons growing in tissue culture94Retinal ganglion cells project to optic tectumThere is a topographic map10Regenerating retinal ganglion neurons project to their appropriate positionAxons know where to goRoger Sperry11normal frog frog with rotated eyeSperry’s Classic Experiment: axons know where to go12Normal frog Frog with rotated eyes5Chemoaffinity HypothesisSpecificity of wiring is based on chemical tags. Individual neurons express distinct molecular markers during development. The formation of appropriate synaptic connections depends on the matching of complementary molecules on pre- and postsynaptic neurons3 tenets of the hypothesis1. neurons are intrinsically different from one another2. Differences in position are biochemical in nature3. Differences are acquired early in development13What are the molecules in the optic tectumthat guide retinal ganglion cells?How is the topographic map of retinal axons established?14Posterior retinal axonsonly grow on anterior tectumAnterior retinal axons grow on bothHow to isolate molecules involved in guidance of retinal axonsDevelop an assay:culture retinal neurons (A or P)with tectum (A and P) “stripe assay”156Ephrins are guidance cues (ligands) in the optic tectumEphrin receptors (receptors) are in the retina axonsMany receptors, many ligands16Ephrin receptors are receptor tyrosine kinases17Ephrin receptors are receptor tyrosine kinasesephrin ligands are tethered to membranesBoth ephrins and Ephrin receptors can activate intracellular signaling===Bidirectional signaling187Boundaries of ephrin A protein restrict growth cones of neurons expressing Ephrin-A receptor. Eph receptors and ephrins restrict growth-cone migration by repulsion19Gradients of Ephrin receptors in the retina andephrins in the tectum set up the topographic mapAnterior retinal axons project to posterior tectum:Low levels Ephrin receptor read only high levels of ephrinPosterior retinal axons project to anterior tectum:High levels Ephrin receptor read low levels of ephrinreceptorligand20Axons Reach their Destination in a Series of Discrete Steps- Axons reach distant targets in a series of discrete steps- Make decisions at frequent intervals along the path- e.g. retinal axon path to the optic tectum- At least 10 steps on the journey218Ganglion cells cross (or not) at the optic chiasmTime course of retinal axon growthSome axons cross(contralateral projections)Others do not cross (ipsilateral projections)22How do axons decide whether or not to cross?Early in development all projections are contralateralAt the time ipsilateral projections are formed, Ephrin B receptors are expressed in posterior (temporal) retinaephrin B becomes expressed in the optic chiasmEphrins act at multiple steps to guide axons to their targetsby steering them away from inappropriate targets23Activation of axon guidance signaling pathwaysinfluences actin polymerization249Principles of Axon Guidance• Chemoaffinity Hypothesis – the specificity of wiring is based on recognition of chemical cues• Axons reach their targets in a series of discrete steps• Different cells respond to the same guidance cues in different ways• Chemical cues exist at many points along the axon guidance pathway e.g the optic tectum and optic tract25Next time: Axons recognize short-range, long-rangeattractive and repulsive