Lecture 11 Axon Pathfinding 02 25 2015 Salamender take neural fold plate and rotate around Mauhner cells found in Rhombomere 4 migrate where they re supposed to go actually in the opposite start along their path think theyre going posterior but realy anterior then they hit boundary and cell surface signals plus morphogens tell them which way to really go if it was completely intrinsic they would keep going but this shows that they respond to environmental cues ablated guidepost go the wrong way Growth cone itself process at end of axon that is testing its environmentmakes al the decisions itself rather than send signal back to ucleus Self autonomous structure Cross section of frog brain eye cup and stuff Connection from eye to optical region from brain Sever connection from retinal neuron and its target If growth cone at end of axon has passed point of where you cut it finds its way in its own even without its body Ramon y Cajal pioneer of neuroanatomy Ross G Harrison father of cell culture father of neuroanatomy Ramon y Cajal discovered things just by looking at tissue sections first to describe that at end of axons there are amoeboid like structures just moving around first to describe structure of growth cone and what they might be doing know who Ramon y Cajal is Ross G Harrison father of cell culture Interested in tissue interactions In order to watch tissue interactions outside the animal he used basic growth medium to use cell culture to look at neurons growing out of an explant Take neural tissue explant put in culture dish and basic medium on it glucose and salt saw axons grow out of the explant Watch grey video First to show that the cll bodies are located iwthin the nervous system spinal cord and send axons out to protrude and the growth cones go and feel out the environment Book confusing to tell which is in vivo or in vitro Figure B Developing frog tail transparengt see early pionerrng axons growth out and observe growth cones Watch video of axon growth cones finding their way and leaving axon behind differences between axons that grow out first and second in nervous system First very arborized growth cones with many processes coming off of pioneer leader axons much more complex growth cones them Second follower axons Less complex a few processes coming off them Follow along the first axons Processes that come off Filopodia finger like projections that feel their way reaching out have receptors to read the environment Lamellipodia flat get feedback from filopodia move the axon along these are the amoeboid like structures See ppt for more images words Usually in vivo transgenic label GFP now many other colors dyes incorporated into plasma membrane of axons DiI DiI non transgenic way of labeling axons it s taken up and diffuses back and you can use it to watch them grow These are the two in vivo methods Growth is at the end of the axon right at the base of the growth cone Growth cone is autonomous adds on units of microtubules at end of axon Flourescently label tubulins make up microtubules so that they only let off so many light units and then it will run out and yo can photobleach make an axon that has fluorescent units at the base right under growth cone and when you fry the middle area and bleach it you can see if the growth happens before or after bleached area see that it happens after by the base of the growth cone OR second image see ppt Put two beads and see if there is growth between the two beads o Yes there is some growth in the middle of the axon but more growth at the end Or pulse a fluorescent label into filopodio it will over time get further and further out showing that the axon is growing there last image https www youtube com watch feature player detailpage v SyJ gz4P Omo make long tubes that can push plasma membrane forward ends up pulling p zone wasn t paying attention learn this c zone once growth cone decides where to go they start to span out if particular filopodia gets positive signal more start growing there and microtubules go there and you get pull towards that ligand whatever is needed in growth cone is brought along microtubules Growth cone uses myosin tether to actually move Actin filament assembly https www youtube com watch feature player detailpage v VVgXDW 8O4U monomer floating around and run into each other added onto the end get negative and and then always added onto positive end This is how groth would ake placein growth cone filopodia wave action removing part of the base watch video https www youtube com watch feature player detailpage v FzcTgrxMzZk t 129 microtubules are like hollow tubes when moving cargo from cell body to growth cone this is how it works see middle of video drugs that either promote polymerization or depolymerize Particular drugs used in classic study cytochalasin depolymerizes once you start to get rid of the actin polymerization the filopodia can t find their way or make it to the next guidepost so they stop moving or they go the wrong way Shows that we need actin polymerization Book switched it around figure is opposite of text Drop beads onto in vitro growing growth cone and it will stick to cell surface the beads end up moving along with motion of plasma membrane If it lands at the end the bead will move backward retrograde flow of actin Add new actin and lose more actin at the base and that s where you get more movement badk towards center of growth cone Retrograde moving backward Labeling axon from growth cone and it moves backward retrograde Anteriograde moving forward By adding drug that stops atcin filaments it stops movement of the bead and you end up stopping the growth of this growth cone If you stop movement of actin polymerization and depolymerization stop growth of growth cone https www youtube com watch feature player detailpage list PLesoAWBu7pfTH 0HEq6aHklD Gctpo k8m v tvFB eOzJs https www youtube com watch feature player detailpage v SyJ gz4P Omo how physically does growth cone actually move Filopodia feel their way out often feeling extracellular matrix molecules n matrix or on another axon they have actin that make contact with it Positive signal something happens actin filaments in contact with myosin microtubules microtubules are pulled along and give motion forward Actin at tips engage cluth o Myosin chains linking microtubules pull axon forward In a dish filopodia are equal onboth sides and front and axon will just grow straight forward Contact with another cell an extracellular