1. Axon Outgrowth and Pathfinding Additional notes

2. Growth Cones Nerve cell processes (both axons and dendrites) grow specialized terminations, called growth cones. This is the site from which neurite extensions arise. Note the appearance in cortex and extensions (lamellopodia, filipodia (finer). These extensions are filled with organelles (e.g., mito, SER, vesicles), because synthesis of new membrane is occurring rapidly.

3. Growth Cones (cont’d) Growth occurs distally (at the end): cytoskeleton, microtubules, filamentous actin and neurofilaments are important components with the “+” ends (i.e., tubulin polarizing) oriented distally. Tubules: provide highway for fast transport of materials where needed (end). Fibriolar actin network: changes shape, which drives the outward movement. (and polymerization).

4. Mechanism of Advance Constant actin polymerization at the edge  push forward with rearward flow of polymerized actin and depolymerization (“caterpillar tread”). This process can occur only on a permissive substratum. These are… - ECM molecules (e.g., laminin-1, fibronectin). - Cell surface molecules (e.g., cadherins). These substratum bind to cell surface receptors (integrins), which link to cellular cytoskeleton.

5. Guidance Cues for Growth Cone Advance and Axon Outgrowth The movement of growth cones is guided by both attractive and repulsive cues, which either activate or inhibit actin polymerization or the attachment of cell surface receptors to this substrata. Two early theories: i.Axons advance more or less randomly and were “fine-tuned” as they reached their proper target (either repelled or attachment strengthened). ii.Axons grow along very pre-set (stereotyped) trajectories and growth is highly directed and precise throughout (not just at the target). The latter has been most supported by research over the last 50+ years.

6. Stepwise Migration How do axons make such a long journey (in some cases, several cm)? Much like the way we tackle the material in this class: breaking the process up into smaller steps. So, the pathway they follow is divided into discrete segments, bounded by guidepost cells, which provide important guidance information. What kind of study may have informed us about the function of these cells?

7. Answer: Ablation studies. In the development of nerve tracts, earlier extending axons (“pioneers”) can serve as a simple scaffold for the rest, simplifying and accelerating the process.

8. Attractive and Repulsive Cues How do axons navigate each segment? Both local and long-range cues exist (analogous to endocrine and paracrine signals), which act in concert e.g., what keeps neural crest cells migratory in the anterior compartment? answer: repulsive cues are sent out from posterior compartment cells “hem in”. What are these repulsive cues? Still an active area of research…examples can be inhibition of actin polymerization or cell surface receptor binding. Note: repulsive cues can be strong enough to Δ the morphology of growth cones  ”growth cone collapse”.

9. Long-range cues: Chemotrophic cues. e.g., chemoattraction: diffusible factors secreted by targets of the depolarizing axon, as in “come hither”. Cortex cell studies have provided much information about this phenomenon; e.g., work with NGF. e.g. of chemorepulsion: a “push-from-behind” or “get away” secreted by a group of cells near the cell body propels the axon away. Chemorepulsion can also deflect axons away from cell groups they should avoid.

10. Molecules Mediating this Guidance 1.Local a. ECM molecules; e.g., laminin-1 – a potent stimulator of axon extension - a “positive corridor” for several axons, as well as for neural crest cells. Others: fibronectin, tenascin. Integrins are the major receptor type, present on axons, which modulate inhibitory effects; e.g., proteoglycans, tenascins (specific signal transducing apparatus of the cell).

11. b. Cell surface molecules from local cells. e.g., those from IgG genes, such as NCAM, bind to specific homologous molecules on the surface of the axon and allow it to adhere and extend. e.g., cadherins 2. Long-range Semaphorins: secreted proteins + transmembrane domains; a repellant that leads to growth cone collapse. Netrins: contributes a chemotropic (attractant) activity, as well as repellant (push from behind) in specific situations. Structure is highly analogous to ECM molecules; so, distinction between long-and short-range cues is not sharp.

12. Honing in on the Final Target: Positional Information Recent active research has revealed that the early theories of gradients of positional information (which we know to be important in establishing A/P polarity in neuronal migration) appear to apply to the formation of the precise topographical patterns of axonal connections with their targets. One candidate: Ephrins (still an active area of research)

13. Growth cones guide axons in the developing nervous system

14. The structure and action of growth cones

15. [Ca 2+ ] intracellular

16. The major classes of axon guidance molecules (secreted and transmembrane)

17. Signals in embryo affect growth cones and growing axons

18. Netrins chemotropically regulate pathway formation in a developing spinal cord wild type netrin knockout

19. Mechanisms of topographic mapping in the vertebrate visual system or Nasal or Temporal

20. Mechanisms of topographic mapping in the vertebrate visual system Retina Tectum (Anterior) (Posterior)

21. Target-derived trophic support regulates survival of related neurons

22. Changes to synapses during early postnatal life in the mammalian PNS

23. Effect of the neurotrophin NGF on the outgrowth of neurites and survival of neurons 8 day chick sensory ganglion without NGF 24 hr after addition of NGF 9 day mouse embryo sympathetic ganglion treated with antibodies to NGF

24. Neurotrophin receptors and their specificity for the neurotrophins

25. Signaling through the neurotrophins and their receptors

26. Hardwiring the Brain: Endocannabinoids Shape Neuronal Connectivity Paul Berghuis, Ann M. Rajnicek, Yury M. Morozov, Ruth A. Ross, Jan Mulder, Gabriella M. Urbán, Krisztina Monory, Giovanni Marsicano, Michela Matteoli, Alison Canty, Andrew J. Irving, István Katona, Yuchio Yanagawa, Pasko Rakic, Beat Lutz, Ken Mackie, Tibor Harkany The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB1 cannabinoid receptors (CB1Rs) are enriched in the axonal growth cones of GABAergic interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB1R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB1Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo. Science 25 May 2007 Vol. 316, pp. 1212 - 1216

27. The Approach Analyze innervation of neurons in vivo in CB 1 R knockout mice Determine the spatial and temporal expression of CB 1 R and endocannabinoids in vivo Examine the effects of CB 1 R agonists and antagonists on neurons in culture Background Axon guidance is crucial to the embryonic wiring of the cerebral cortex G protein-coupled CB 1 cannabinoid receptors respond to marijuana and to endocannabinoids in the adult Endocannabinoids released from postsynaptic cells retrogradely suppress transmitter release from presynaptic cells in adults CB 1 R are expressed on developing axonal projections, but whether endocannabinoids are axon guidance molecules was unknown

28. Figure 1 Temporal and spatial coincidence of CB 1 R localization with endocannabinoid availability during corticogenesis 7 µm hippocampus cerebral cortex pyramidal neurons GABAergic interneurons hippocampus RECE P TORSRECE P TORS LIGANDSLIGANDS GABAergic interneuronspyramidal neurons DAGL  and NAPE-PLD are synthetic enzymes for endocannabinoids --------------- cerebral cortex ---------------

29. Figures 2&3 Agonists induce CB 1 R removal from filopodia of GABAergic neurons in culture and cause repulsive turning WIN 55,212-2 is a CB 1 R agonist AM251 is a CB 1 R antagonist control AEA is a CB 1 R agonist

30. Physiological importance of CB 1 R-mediated growth cone repulsion: gene knockout alters distribution and density of inhibitory innervation wild type knockout

31. Hardwiring the Brain: Endocannabinoids Shape Neuronal Connectivity Paul Berghuis, Ann M. Rajnicek, Yury M. Morozov, Ruth A. Ross, Jan Mulder, Gabriella M. Urbán, Krisztina Monory, Giovanni Marsicano, Michela Matteoli, Alison Canty, Andrew J. Irving, István Katona, Yuchio Yanagawa, Pasko Rakic, Beat Lutz, Ken Mackie, Tibor Harkany Science 25 May 2007 Vol. 316, pp. 1212 - 1216 Conclusions: 1) Endocannabinoids and their receptors are in the right place in vivo, they affect growth cone turning in vitro, and they affect subsequent innervation in vivo. 2) Endocannabinoid signaling regulates growth cone steering and thus axon guidance. 3) Prenatal exposure to cannabis could affect neuronal development and account for cognitive and behavioral deficits.