腦部是一個由眾多神經元網路所構成之多功能且複雜精細的系統；神經元間借由特殊的細胞連結--突觸-進行溝通。每個神經元送出軸突，行進相當距離，然後和適當的目標形成突觸。軸突的生長和突觸的形成，乃是受存於細胞表面，或細胞外環境的眾多蛋白質所調控。為研究此眾多蛋白質對軸突生長和突觸形成的影響。一個以晶片為基礎的系統被開發出來。這一系統含數個微圖案所形成區域。神經元被培養於一區域，而軸突則生長至另遠端另一區域。在這遠端區域，軸突生長及可釋放之突觸囊泡—前突觸形成的標誌，皆可被量化。
在此系統中，可將各種蛋白質塗覆於特定區域上，使軸突暴露於該種蛋白質。藉著測量軸突生長及可釋放之突觸囊泡的數量，用以研究與比較各種蛋白質的效能，。我利用此系統研究neuroligin 1、laminin和collagen對軸突生長及前突觸形成的效能。亦可將單一蛋白質塗覆於不同區域表面，使神經細胞不同部份暴露於該種蛋白質。藉著測量在此條件下前突觸形成及軸突的數量。藉以研究該種蛋白質引發信號的有效傳遞範圍。以此種方法被研究Neuroligin 1顯示其對前突觸形成及軸突成長的信號傳遞，是屬於短距離局部的作用。

The brain is a versatile system and consists of an intricate network of neurons that communicate with each other through special cell contact sites- synapses. Each neuron sends its axon to travel long distance, to meet appropriate targets and to make synapses. The axonal outgrowth and synaptogenesis are regulated by various proteins which exist on cells surface or in the extracellular environments. For investigating the effects of various exogenous proteins on axonal outgrowth and pre-synaptic specification, a chip-based system has been developed. The system comprises of multiple micropatterned areas mounted on the surface of a glass chip. Neurons are cultured on one area on the chip with their axons being guided to grow in another area on the same surface. The amount of axons and of the clusters of releasing-competent synaptic vesicles (RSV), a marker of pre-synaptic specification, in this latter area can be quantified.
In this system, various proteins can be coated to a specific area where axons grow. By quantifying the different amounts of axons and pre-synaptic specification in this specific area, the effects of different proteins can be compared and investigated. The effects of neuroligin 1, laminin, and collagen on axonal outgrowth as well as pre-synaptic specification have been studied here by using this system. A protein can also be coated at varied areas, and thereby different regions of neurons are exposed to this single protein. The amounts of axons and pre-synaptic specification under such conditions can also be quantified. Thus, the effective signal range of a specific protein that could travel within a neuron can be investigated. By using this method, neurolign1 has been studied and demonstrated to mediate pre-synaptic specification within short ranges.

VI. REFERENCES
Ackerman, S. L., L. P. Kozak, S. A. Przyborski, L. A. Rund, B. B. Boyer and B. B. Knowles (1997). "The mouse rostral cerebellar malformation gene encodes an UNC-5-like protein." Nature 386(6627): 838-842.
Ahmari, S. E., J. Buchanan and S. J. Smith (2000). "Assembly of presynaptic active zones from cytoplasmic transport packets." Nat Neurosci 3(5): 445-451.
Aumailley, M., L. Bruckner-Tuderman, W. G. Carter, R. Deutzmann, D. Edgar, P. Ekblom, J. Engel, E. Engvall, E. Hohenester, J. C. R. Jones, H. K. Kleinman, M. P. Marinkovich, G. R. Martin, U. Mayer, G. Meneguzzi, J. H. Miner, K. Miyazaki, M. Patarroyo, M. Paulsson, V. Quaranta, J. R. Sanes, T. Sasaki, K. Sekiguchi, L. M. Sorokin, J. F. Talts, K. Tryggvason, J. Uitto, I. Virtanen, K. von der Mark, U. M. Wewer, Y. Yamada and P. D. Yurchenco (2005). "A simplified laminin nomenclature." Matrix Biology 24(5): 326-332.
Betz, W., F. Mao and G. Bewick (1992). "Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals." The Journal of Neuroscience 12(2): 363-375.
Betz, W. J., F. Mao and C. B. Smith (1996). "Imaging exocytosis and endocytosis." Current Opinion in Neurobiology 6(3): 365-371.
Boucard, A. A., A. A. Chubykin, D. Comoletti, P. Taylor and T. C. Südhof (2005). "A Splice Code for trans-Synaptic Cell Adhesion Mediated by Binding of Neuroligin 1 to α- and β-Neurexins." Neuron 48(2): 229-236.
Brakebusch, C. and R. Fassler (2003). "The integrin-actin connection, an eternal love affair." The EMBO journal 22(10): 2324-2333.
Bridgman, P. C. and M. E. Dailey (1989). "The organization of myosin and actin in rapid frozen nerve growth cones." The Journal of Cell Biology 108(1): 95-109.
Brose, K., K. S. Bland, K. H. Wang, D. Arnott, W. Henzel, C. S. Goodman, M. Tessier-Lavigne and T. Kidd (1999). "Slit Proteins Bind Robo Receptors and Have an Evolutionarily Conserved Role in Repulsive Axon Guidance." Cell 96(6): 795-806.
Buck, K. B. and J. Q. Zheng (2002). "Growth Cone Turning Induced by Direct Local Modification of Microtubule Dynamics." The Journal of Neuroscience 22(21): 9358-9367.
Burnette, D. T., A. W. Schaefer, L. Ji, G. Danuser and P. Forscher (2007). "Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones." Nat Cell Biol 9(12): 1360-1369.
Charron, F., E. Stein, J. Jeong, A. P. McMahon and M. Tessier-Lavigne (2003). "The Morphogen Sonic Hedgehog Is an Axonal Chemoattractant that Collaborates with Netrin-1 in Midline Axon Guidance." Cell 113(1): 11-23.
Chen, W.-S., O. B. Villaflores, C.-F. Lu, H.-I. Wu, Y.-J. Chen, C.-Y. Teng, Y.-C. Chang, S.-L. Chang and T.-Y. Wu (2012). "Functional expression of rat neuroligin-1 extracellular fragment by a bi-cistronic baculovirus expression vector." Protein Expression and Purification 81(1): 18-24.
Chen, Z.-L., V. Haegeli, H. Yu and S. Strickland (2009). "Cortical deficiency of laminin γ1 impairs the AKT/GSK-3β signaling pathway and leads to defects in neurite outgrowth and neuronal migration." Developmental Biology 327(1): 158-168.
Cheng, H.-H., S.-H. Liu, H.-C. Lee, Y.-S. Lin, Z.-H. Huang, C.-I. Hsu, Y.-C. Chen and Y.-C. Chang (2006). "Heavy chain of cytoplasmic dynein is a major component of the postsynaptic density fraction." Journal of Neuroscience Research 84(2): 244-254.
Cheng, W.-Y., W.-L. Hsu, H.-H. Cheng, Z.-H. Huang and Y.-C. Chang (2009). "An observation chamber for studying temperature-dependent and drug-induced events in live neurons using fluorescence microscopy." Analytical Biochemistry 386(1): 105-112.
Chih, B., H. Engelman and P. Scheiffele (2005). "Control of Excitatory and Inhibitory Synapse Formation by Neuroligins." Science 307(5713): 1324-1328.
Chih, B., L. Gollan and P. Scheiffele (2006). "Alternative Splicing Controls Selective Trans-Synaptic Interactions of the Neuroligin-Neurexin Complex." Neuron 51(2): 171-178.
Chilton, J. K. (2006). "Molecular mechanisms of axon guidance." Developmental Biology 292(1): 13-24.
Craig, A. M., E. R. Graf and M. W. Linhoff (2006). "How to build a central synapse: clues from cell culture." Trends in Neurosciences 29(1): 8-20.
Craig, A. M. and Y. Kang (2007). "Neurexin–neuroligin signaling in synapse development." Current Opinion in Neurobiology 17(1): 43-52.
Da Silva, J. S., M. Medina, C. Zuliani, A. Di Nardo, W. Witke and C. G. Dotti (2003). "RhoA/ROCK regulation of neuritogenesis via profilin IIa–mediated control of actin stability." The Journal of Cell Biology 162(7): 1267-1279.
Dalva, M. B., M. A. Takasu, M. Z. Lin, S. M. Shamah, L. Hu, N. W. Gale and M. E. Greenberg (2000). "EphB Receptors Interact with NMDA Receptors and Regulate Excitatory Synapse Formation." Cell 103(6): 945-956.
Dean C. and Dresbach T. (2006). "Neuroligins and neurexins: linking cell adhesion, synapse formation and cognitive function." Trens Neurosci. 29(1):21-9.
Dean, C., F. G. Scholl, J. Choih, S. DeMaria, J. Berger, E. Isacoff and P. Scheiffele (2003). "Neurexin mediates the assembly of presynaptic terminals." Nat Neurosci 6(7): 708-716.
Dickson, B. J. (2002). "Molecular Mechanisms of Axon Guidance." Science 298(5600): 1959-1964.
Dotti, C., C. Sullivan and G. Banker (1988). "The establishment of polarity by hippocampal neurons in culture." The Journal of Neuroscience 8(4): 1454-1468.
Drees, F. and F. B. Gertler (2008). "Ena/VASP: proteins at the tip of the nervous system." Current Opinion in Neurobiology 18(1): 53-59.
Forscher, P. and S. J. Smith (1988). "Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone." The Journal of Cell Biology 107(4): 1505-1516.
Freire, E., F. C. A. Gomes, R. Linden, V. M. Neto and T. Coelho-Sampaio (2002). "Structure of laminin substrate modulates cellular signaling for neuritogenesis." Journal of Cell Science 115(24): 4867-4876.
Friedman, H. V., T. Bresler, C. C. Garner and N. E. Ziv (2000). "Assembly of New Individual Excitatory Synapses: Time Course and Temporal Order of Synaptic Molecule Recruitment." Neuron 27(1): 57-69.
Goldberg, D. J. and D. W. Burmeister (1986). "Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy." The Journal of Cell Biology 103(5): 1921-1931.
Goley, E. D. and M. D. Welch (2006). "The ARP2/3 complex: an actin nucleator comes of age." Nat Rev Mol Cell Biol 7(10): 713-726.
Goode, B. L. and M. J. Eck (2007). "Mechanism and Function of Formins in the Control of Actin Assembly." Annual Review of Biochemistry 76(1): 593-627.
Grabham, P. W., G. E. Seale, M. Bennecib, D. J. Goldberg and R. B. Vallee (2007). "Cytoplasmic Dynein and LIS1 Are Required for Microtubule Advance during Growth Cone Remodeling and Fast Axonal Outgrowth." The Journal of Neuroscience 27(21): 5823-5834.
Graf, E. R., Y. Kang, A. M. Hauner and A. M. Craig "Structure Function and Splice Site Analysis of the Synaptogenic Activity of the Neurexin-1β LNS Domain." The Journal of Neuroscience 26(16): 4256-4265.
Graf, E. R., X. Zhang, S.-X. Jin, M. W. Linhoff and A. M. Craig (2004). "Neurexins Induce Differentiation of GABA and Glutamate Postsynaptic Specializations via Neuroligins." Cell 119(7): 1013-1026.
Grifman, M., N. Galyam, S. Seidman and H. Soreq (1998). "Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis." Proceedings of the National Academy of Sciences 95(23): 13935-13940.
Hong, K., L. Hinck, M. Nishiyama, M.-m. Poo, M. Tessier-Lavigne and E. Stein (1999). "A Ligand-Gated Association between Cytoplasmic Domains of UNC5 and DCC Family Receptors Converts Netrin-Induced Growth Cone Attraction to Repulsion." Cell 97(7): 927-941.
Julien, F., A. Bechara, R. Fiore, H. Nawabi, H. Zhou, C. Hoyo-Becerra, M. Bozon, G. Rougon, M. Grumet, A. W. Püschel, J. R. Sanes and V. Castellani (2005). "Dual Functional Activity of Semaphorin 3B Is Required for Positioning the Anterior Commissure." Neuron 48(1): 63-75.
Kannenberg, K., W. Sieghart and H. Reuter (1999). "Clusters of GABAA receptors on cultured hippocampal cells correlate only partially with functional synapses." European Journal of Neuroscience 11(4): 1256-1264.
Kaplan, D. R. and F. D. Miller (2000). "Neurotrophin signal transduction in the nervous system." Current Opinion in Neurobiology 10(3): 381-391.
Keino-Masu, K., M. Masu, L. Hinck, E. D. Leonardo, S. S. Y. Chan, J. G. Culotti and M. Tessier-Lavigne (1996). "Deleted in Colorectal Cancer (DCC) Encodes a Netrin Receptor." Cell 87(2): 175-185.
Kennedy, T. E., T. Serafini, J. de la Torre and M. Tessier-Lavigne (1994). "Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord." Cell 78(3): 425-435.
Kennedy, T. E., H. Wang, W. Marshall and M. Tessier-Lavigne (2006). "Axon Guidance by Diffusible Chemoattractants: A Gradient of Netrin Protein in the Developing Spinal Cord." The Journal of Neuroscience 26(34): 8866-8874.
Kidd, T., K. S. Bland and C. S. Goodman (1999). "Slit Is the Midline Repellent for the Robo Receptor in Drosophila." Cell 96(6): 785-794.
Kidd, T., K. Brose, K. J. Mitchell, R. D. Fetter, M. Tessier-Lavigne, C. S. Goodman and G. Tear (1998). "Roundabout Controls Axon Crossing of the CNS Midline and Defines a Novel Subfamily of Evolutionarily Conserved Guidance Receptors." Cell 92(2): 205-215.
Kitsukawa, T., M. Shimizu, M. Sanbo, T. Hirata, M. Taniguchi, Y. Bekku, T. Yagi and H. Fujisawa (1997). "Neuropilin–Semaphorin III/D-Mediated Chemorepulsive Signals Play a Crucial Role in Peripheral Nerve Projection in Mice." Neuron 19(5): 995-1005.
Kneussel, M. and H. Betz (2000). "Receptors, gephyrin and gephyrin-associated proteins: novel insights into the assembly of inhibitory postsynaptic membrane specializations." The Journal of Physiology 525(1): 1-9.
Koester, M. P., O. Müller and G. E. Pollerberg (2007). "Adenomatous Polyposis Coli Is Differentially Distributed in Growth Cones and Modulates Their Steering." The Journal of Neuroscience 27(46): 12590-12600.
Lee, C.-H., T. Herman, T. R. Clandinin, R. Lee and S. L. Zipursky (2001). "N-Cadherin Regulates Target Specificity in the Drosophila Visual System." Neuron 30(2): 437-450.
Leonardo, E. D., L. Hinck, M. Masu, K. Keino-Masu, S. L. Ackerman and M. Tessier-Lavigne (1997). "Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors." Nature 386(6627): 833-838.
Lin, C. H., E. M. Espreafico, M. S. Mooseker and P. Forscher (1996). "Myosin Drives Retrograde F-Actin Flow in Neuronal Growth Cones." Neuron 16(4): 769-782.
Lowery L.A. and Van Vactor D (2009). " The trip of the tip: understanding the growth cone machinery." Nat Rev Mol Cell Biol. 10(5): 332–343.
Luo, Y., D. Raible and J. A. Raper (1993). "Collapsin: A protein in brain that induces the collapse and paralysis of neuronal growth cones." Cell 75(2): 217-227.
Lyuksyutova, A. I., C.-C. Lu, N. Milanesio, L. A. King, N. Guo, Y. Wang, J. Nathans, M. Tessier-Lavigne and Y. Zou (2003). "Anterior-Posterior Guidance of Commissural Axons by Wnt-Frizzled Signaling." Science 302(5652): 1984-1988.
Ma, L. and M. Tessier-Lavigne (2007). "Dual Branch-Promoting and Branch-Repelling Actions of Slit/Robo Signaling on Peripheral and Central Branches of Developing Sensory Axons." The Journal of Neuroscience 27(25): 6843-6851.
Mann, F., S. Ray, W. A. Harris and C. E. Holt (2002). "Topographic Mapping in Dorsoventral Axis of the Xenopus Retinotectal System Depends on Signaling through Ephrin-B Ligands." Neuron 35(3): 461-473.
McLaughlin, T., R. Hindges, P. A. Yates and D. D. M. O'Leary (2003). "Bifunctional action of ephrin-B1 as a repellent and attractant to control bidirectional branch extension in dorsal-ventral retinotopic mapping." Development 130(11): 2407-2418.
Missler, M., W. Zhang, A. Rohlmann, G. Kattenstroth, R. E. Hammer, K. Gottmann and T. C. Sudhof (2003). "[alpha]-Neurexins couple Ca2+ channels to synaptic vesicle exocytosis." Nature 423(6943): 939-948.
O’Brien, R. J., D. Xu, R. S. Petralia, O. Steward, R. L. Huganir and P. Worley (1999). "Synaptic Clustering of AMPA Receptors by the Extracellular Immediate-Early Gene Product Narp." Neuron 23(2): 309-323.
Okabe, S., A. Miwa and H. Okado (2001). "Spine Formation and Correlated Assembly of Presynaptic and Postsynaptic Molecules." The Journal of Neuroscience 21(16): 6105-6114.
Plantman, S., M. Patarroyo, K. Fried, A. Domogatskaya, K. Tryggvason, H. Hammarberg and S. Cullheim (2008). "Integrin-laminin interactions controlling neurite outgrowth from adult DRG neurons in vitro." Molecular and Cellular Neuroscience 39(1): 50-62.
Prakash, S., J. C. Caldwell, D. F. Eberl and T. R. Clandinin (2005). "Drosophila N-cadherin mediates an attractive interaction between photoreceptor axons and their targets." Nat Neurosci 8(4): 443-450.
Raper, J. A. (2000). "Semaphorins and their receptors in vertebrates and invertebrates." Current Opinion in Neurobiology 10(1): 88-94.
Rizzoli, S. O. and W. J. Betz (2005). "Synaptic vesicle pools." Nat Rev Neurosci 6(1): 57-69.
Schaefer, A. W., N. Kabir and P. Forscher (2002). "Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones." The Journal of Cell Biology 158(1): 139-152.
Schaefer, A. W., V. T. Schoonderwoert, L. Ji, N. Mederios, G. Danuser and P. Forscher (2008). "Coordination of actin filament and microtubule dynamics during neurite outgrowth." Developmental cell 15(1): 146-162.
Scheiffele, P. (2003). "CELL-CELL SIGNALING DURING SYNAPSE FORMATION IN THE CNS." Annual Review of Neuroscience 26(1): 485-508.
Scheiffele, P., J. Fan, J. Choih, R. Fetter and T. Serafini (2000). "Neuroligin Expressed in Nonneuronal Cells Triggers Presynaptic Development in Contacting Axons." Cell 101(6): 657-669.
Shen, K. and C. I. Bargmann (2003). "The Immunoglobulin Superfamily Protein SYG-1 Determines the Location of Specific Synapses in C. elegans." Cell 112(5): 619-630.
Shen, K., R. D. Fetter and C. I. Bargmann (2004). "Synaptic Specificity Is Generated by the Synaptic Guidepost Protein SYG-2 and Its Receptor, SYG-1." Cell 116(6): 869-881.
Soekarno, A., B. Lom and P. E. Hockberger (1993). "Pathfinding by Neuroblastoma Cells in Culture Is Directed by Preferential Adhesion to Positively Charged Surfaces." NeuroImage 1(2): 129-144.
Song, J.-Y., K. Ichtchenko, T. C. Südhof and N. Brose (1999). "Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses." Proceedings of the National Academy of Sciences 96(3): 1100-1105.
Suter, D. M., A. W. Schaefer and P. Forscher (2004). "Microtubule Dynamics Are Necessary for Src Family Kinase-Dependent Growth Cone Steering." Current Biology 14(13): 1194-1199.
Tamagnone, L. and P. M. Comoglio (2000). "Signalling by semaphorin receptors: cell guidance and beyond." Trends in Cell Biology 10(9): 377-383.
Tanaka, E., T. Ho and M. W. Kirschner (1995). "The role of microtubule dynamics in growth cone motility and axonal growth." The Journal of Cell Biology 128(1): 139-155.
Timpl, R. (1996). "Macromolecular organization of basement membranes." Current Opinion in Cell Biology 8(5): 618-624.
Trousse, F., E. Martí, P. Gruss, M. Torres and P. Bovolenta (2001). "Control of retinal ganglion cell axon growth: a new role for Sonic hedgehog." Development 128(20): 3927-3936.
Wang, K. H., K. Brose, D. Arnott, T. Kidd, C. S. Goodman, W. Henzel and M. Tessier-Lavigne (1999). "Biochemical Purification of a Mammalian Slit Protein as a Positive Regulator of Sensory Axon Elongation and Branching." Cell 96(6): 771-784.
Washbourne, P., J. E. Bennett and A. K. McAllister (2002). "Rapid recruitment of NMDA receptor transport packets to nascent synapses." Nat Neurosci 5(8): 751-759.
Wu, H.-I., G.-H. Cheng, Y.-Y. Wong, C.-M. Lin, W. Fang, W.-Y. Chow and Y.-C. Chang (2010). "A lab-on-a-chip platform for studying the subcellular functional proteome of neuronal axons." Lab on a Chip 10(5): 647-653.
Yamagata, M., J. R. Sanes and J. A. Weiner (2003). "Synaptic adhesion molecules." Current Opinion in Cell Biology 15(5): 621-632.
Yamagata, M., J. A. Weiner and J. R. Sanes (2002). "Sidekicks: Synaptic Adhesion Molecules that Promote Lamina-Specific Connectivity in the Retina." Cell 110(5): 649-660.
Yamauchi, K., K. D. Phan and S. J. Butler (2008). "BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions." Development 135(6): 1119-1128.
Yoshikawa, S., R. D. McKinnon, M. Kokel and J. B. Thomas (2003). "Wnt-mediated axon guidance via the Drosophila Derailed receptor." Nature 422(6932): 583-588.
Yoshimura, T., Y. Kawano, N. Arimura, S. Kawabata, A. Kikuchi and K. Kaibuchi (2005). "GSK-3β Regulates Phosphorylation of CRMP-2 and Neuronal Polarity." Cell 120(1): 137-149.
Zhai, R. G., H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv and C. C. Garner (2001). "Assembling the Presynaptic Active Zone: A Characterization of an Active Zone Precursor Vesicle." Neuron 29(1): 131-143.
Zhou, Y., R.-A. F. Gunput and R. J. Pasterkamp (2008). "Semaphorin signaling: progress made and promises ahead." Trends in Biochemical Sciences 33(4): 161-170.