BISC 421 1st Edition Lecture 36 Outline of Current LectureI. Construction of Neural Circuits 2Current LectureConstruction of Neural Circuits 2 Long distance attraction - netrins! The system: chick spinal cord. What attractsRPcommisural neurons to the floorplate?•This has been studied at the spinal cord of a chick •Can label these neurons as they project down to the bottom •No one knows exactly which circuit they are participating in•Convenient to use a chick because the chicks develop outside the mother in the egg-‐ can watch the embryo as it develops-‐ easy to manipulate•The question we are now focusing on-‐ what is attracting these neurons from the roof plate down to the floor plate?Long distance attraction - netrins!The experiment: Isolated the active component from extract of the floorplate (FP) thatinduces growth of the roofplate (RP) to FP. -Molecule identified called Netrin.-Netrin was the first example of a long range attractive cue.•Studied by taking out a ball of neurons from the roof plate and put another extractof the floor plate and see if you can still retain this growth•Find that the axons form the roof plate are extending towards the floor plate so there must be something in the floor plate that attracts the roof plate•Take from this extract, put through biochemical purification steps to figure out the protein that is directing growth of axons•Whatever was directing these axons was also found in the brain of a developing chick (netrin)-‐found this by isolating it from 25000 chicks•Shows that this molecule in this kind of assay could direct activity of these roof plate neurons-‐so let's test knock out netrin: what happens?Netrin knockout!Axon projections from dorsal spinal cord to ventral spinal cord are disrupted. From Serafini et al, Cell 1996• With the knockout of netrin can see that the axons never reach the floor plate and just wander aimlessly.Long distance attraction - netrins!Receptors for Netrin are DCC and UNC5 different subunit combinations mediate either attraction or repulsion •Netrin binds to a receptor (complicated)-‐ just keep in mind that Netrin can have 2 different types of receptors (DCC and UNC5)•When netrin binds to DCC it will cause the axon to be attracted to whatever is releasing netrin •When netrin binds to UNC5, axon will be repelled from netrin secreting moleculeLong distance cues: Netrins and Slit!•Netrin is both attractive and repulsive•There is another receptor called slit that has its own receptor called robo (repulsive)•Signaling not well understood-‐ involves changes in intracellular second messengers •Change in how the cytoskeleton of the cell is active is what we do knowGuidance cues! Attraction: short range: Collagen long range: Netrins Repulsion: short range - Ephrinslong range –Slit, Netrin.•Summary of our idea of how a cell is guided to its targetto cross or not to cross!•Cell being attracted towards the midline and doesn't go up until it crosses the midline-‐ some attractive cue making it go up but only afer it crosses. How does this happen?Changes in expression of growth factors uponmidline crossing! How are axons that are initially attracted to the midline, able to overcome this attraction and cross?Answer: Expression of receptors for guidance factors change as the axon extendstowards the midline.•Think that the axons are initially attracted to the midline and then they become less attracted and then ascend upwards•Once in the floor plate, turn off attraction to netrin and turning on repellent cue slit•DCC turns off and slit (robo) turned on•Temporal control of expression of receptors for guidance cuesMidline crossing – slit repulsion is requiredwild-type Axons slit mutant Nocross once and repulsion: Axonsleave midline stay in midline•Knock out experiment in fly-‐ knock out slit•Find that in the knock out the repulsion is not turned on and therefore the axons stay where they are at the midline and never cross•They still ascend thoughGrowth cones are dynamicMauthner axon – (zebrafish)Jontes, J.D., Buchanan, J. and Smith, S.J (2000)Nature Neuroscience 3: 231-237.•End of the axonThe growth cone!Ramon y Cajal (1890s)Envisioned that the growth cone was “endowed with exquisite chemical sensitivity, rapid ameboid movements and a certain motive force thanks to which it is able to proceed forwardand overcome obstacles in its way…until it reaches its destination."•Without even being able to observe the growth cone-‐ just hypothesized that there was oneStructure of a growth cone!• Forscher, P. and Smith, S.J. (1988) J. Cell Biol. 107: 1505-1516 "•Lamellapodium (http:// ww.neuron.org/cgi/ content/full/17/1/91/ DC1) Movement of the growth cone: polymerization of actin and tubulin! Lamellapodium is tubulin richFilopodia are actin richPolymerization and depolimerization control dynamicsCa2+ regulates dynamics•Actin that has polymerized into tubes which holds the support for lamellapodium•Formation of this actin is dependent on a lot of factors including calcium •Polymerization causes the actin to move forward and depolymerization causes it to retractCalcium is elevated in response to attractant• Localized Ca2+ signals in the growth cone induced by an extracellular gradient of the guidance cue netrin-1• cultured Xenopus spinal neuron injected with the Ca2+-sensitivefluorescence indicator•Evidence that calcium could be one of the signals that is directing the growth coneGrowth cone!• A dynamic structure"• Responds to axon guidance cues with elevation of calcium elevation "• Turing and elongation depend on actin dynamics"The matching problem:•Getng all of these cells dividing but in the end you want the cells in one area of the brain to connect to another area-‐ need the correct number of preaynaptic cells to match up to the correct number of post synaptic cells•How do they know to make the right number•How are the presynaptic cells and postsynaptic cells matched? Don't need to be 1:1 •Simplest system in neuromuscular junction• Other signaling pathways undoubtedly regulate growth cone turning and elongation."•Targets contain a survival signal •What will happen if we remove the limb bud to the number of the motor neurons we have in the spinal cord?•If we remove the limb cord find that on this side of the spinal cord, there are not as many