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研究生(外文):Chin-He Chang
論文名稱(外文):Investigation of the role of glial cells in the synchronous firing and calcium wave propagations in rat cortical neural cultures
指導教授(外文):Chi-Keung ChanDaisy L. Hung
外文關鍵詞:calcium wavephotolysissynchronous firingastrocytesglial cell
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「自發性神經同步活動」在哺乳類神經系統中是個普遍的現象,而這樣的現象對於神經細胞的發育、突觸(synapse)的形成,扮演著極重要的角色。事實上,哺乳類動物的大腦中除了神經細胞之外,佔大多數的卻是神經膠細胞(glial cell)。隨著近年來大量的研究結果顯示,神經膠細胞除了傳統上認為支持、提供養分的角色之外,還參與了神經細胞的許多活動,同時亦可以擔任調節突觸活動的功能。甚至還有證據認為神經膠細胞在突觸的連結與形成的機制上,扮演著舉足經重的角色,這樣的結果的確隱含著神經膠細胞有著直接影響神經可塑性(plasticity)與高等認知功能的緊密關係。由於神經細胞要達到同步化,必要的條件是細胞與細胞之間的連結數目要夠多,意即細胞之間的突觸要達到某一程度的連結。由於最近的研究顯示,神經膠細胞和神經細胞的突觸之間有著緊密的連結,並且參與了活動的調節,因此我們推論神經膠細胞不僅能提供神經細胞養分,同時在自發性神經同步活動中,也可能扮演著重要的作用。
Glial cells are traditionally thought to play the peripheral and supportive role in the CNS (Central Nervous System). However, recent studies are shown that glia have a variety of functions in the synaptic transmission. Therefore, recognition of the importance of glial cells in nervous system functions is increasing, especially regarding the modulation of neural activity.
Synchronized spontaneous Ca2+ spikes in neuronal network cultures represent periodic bursting, which are believed to play a major role during the development of the CNS. It is known that glia, especial astrocytes, are the most abundant cell-type in the CNS, which will also release neurotransmitters. How the neuronal network are shaped and modulated by the glia remain to be studied. In this work, we study the role of glial cells in spontaneous activities by monitoring changes in [Ca2+]i with fluorescence dye. The result displays that the neuronal synchronized firing (SF) remains to be observed under the glia-suppressed cultures (GSCs). However, it can be seen that there are the different patterns in the cultures with the different number of glial cells. These studies demonstrate that glia play an important role in the neuronal network activity during development.
Furthermore, photolysis by using a UV laser is used to create intercellular Ca2+ waves to investigate the effects of glia on synaptic transmission. It is shown that the velocity of Ca2+ wave propagation in the glia-enhanced cultures (GECs) is slower than that of in the GSCs.
Abstract Ⅰ
Contents Ⅱ
List of figures Ⅲ
List of tables Ⅴ

1 Chapter 1 introduction 1

1.1 Synchronization phenomenon in the natural world 1
1.2 Synchronized activities of neural network in the central nervous system 1
1.3 The relationship between synchronous firing 4
1.4 Basic neurobiology of neuron-glia interactions 5
1.5 Do glial cells participate in synchronous firing and epilepsy? 8
1.6 The role of astrocytic calcium waves 11
1.7 The underlying effect of glial cells on the cognitive functions 13
1.8 The approaches and the purposes 15

2 Chapter 2 Experimental setup and procedures 16

Overview 16

2.1 Sample preparations 16
2.1.1 Neuron-glia cocultures (NGCs) 17
2.1.2 Pure astrocytic cultures and preparation of glia-conditioned medium (GCM) 17
2.1.3 Glia-suppressed cultures (GSCs) 18 GSCs with the GCM 18 GSCs in NB/B27 medium 18
2.1.4 Glia-enhanced cultures (GECs) 19 Pre-plated astrocytes 19 Post-plated astrocytes 19

2.2 Imaging system 20
2.2.1 Microscope 20
2.2.2 Optics set-up 21
2.2.3 Temperature controller 22
2.2.4 Intensified Charge-Coupled Device (ICCD) video camera 22
2.2.5 Automation 23

2.3 The UV laser photolysis 23
2.3.1 Fluorescence calcium indicator 23
2.3.2 Pulsed UV laser machaine 24
2.3.3 Flash photolysis-induced calcium waves 24

2.4 Immunocytochemistry 26

3 Chapter 3 Results 27

3.1 Identification of neurons and astroglia in the primary cultures 27
3.2 The different SF patterns of three types of cultures during development 38
3.3 Calcium waves measurements in the three cultured conditions 46
3.4 UV laser-induced Ca2+ propagations induce the neuronal rapid Ca2+ oscillations in the glia-free cultures 47
3.5 Laser-induced the astrocytes wavy-like Ca2+ oscillations 48

4 Chapter 4 discussion 50
4.1 The manipulation of glial cells on the cultures during development 50
4.2 The amount of glial cells influences the neuronal Sf (SF frequency) 53
4.3 The role of astroglia in the Ca2+ waves propagation 54

5 Chapter 5 summary and conclusions 59

6 Reference 60

7 Appendix A 65

8 Appendix B 67

Alvarez-Maubecin V, Garcia-Hernandez F, Williams JT, Van Bocjstaele EJ (2000) Functional coupling between neurons and glia. J Neurosci 20:4091-4098.

Angulo MC, Kozlov AS, Charpak S, Audinat E (2004) Glutamate released from glial cells synchronizes neuronal activity in the hippocampus. J Neurosci 24:6920-6927.

Araque A, Parpura V, Sanzgiri RP, Haydon PG (1999) Tripartite synapses: glia, the unacknowledged partner. Trends Neurosci 22:208-215.

Brewer GJ, Torricelli JR, Evege EK, Price PJ (1993) Optimized survival of hippocampal neurons in B27-supplemented Neurobasl, a new serum-free medium combination. J Neurosci Res 35:567-576.

Burgand EC, Hablitz JL (1994) Development changes in NMDA and non-NMDA receptor-mediated synaptic potentials in rat neocortex. J Neurophysiol 69:230-240.

Chapman B, Stryker MP (1993) Development of orientation selectivity in ferret visual cortex and effects of deprivation. J Neurosci 13:5251-5262.

Charles AC, Merrill JE, Dirksen ER, Sanderson MJ (1991) Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanism stimulation and glutamate. Neuron 6:983-992.

Charles A, Naus C, Zhu D, Kidder G, Dirksen E, Sanderson M (1992) Intercellular calcium signaling via gap junctions in glioma cells. J Cell Biol 118:195-201.

Charles A (2005) Reaching out beyond the synapse: glial intercellular waves coordinate metabolism. Sci STKE 270:1-4.

Connors BW (1984) Initiation of synchronized neuronal bursting in neocortex. Nature 310:685-687.
Cornell-Bell AH, Finkeiner SM, Cooper MS, Smith SJ (1990) Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science 247:470-473.
McCarthy and De Vellis (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85:890-902.

Escobar AL, Cifuentes F, Vergara JL (1995) Detection of Ca2+-transients elicited by flash photolysis of DM-nitrophen with a fast calcium indicator. FERS Lett 364:335-338.

Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG, Carmignoto G (2004) Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43:729-743.

Fields RD, Stevens-Graham B (2002) New insights into neuron-glia communication. Science 298:556-562.

Fields RD (2004) The other half of the brain. Sci Am 290: 54-61.

Guthrie PB, Knappenberger J, Segael M, Bennett MVL, Charles AC (1999) ATP released from astrocytes mediated glial calcium waves. J Neurosci 19:520-528.

Hassigner TD, Atkison PB, Strecker GJ, Whalen LR, Dudek FE, Kossel AH, Kater SB (1995) Evidence for glutamate-mediated activation of hippocampal neurons by glial calcium waves. J Neurobiol 28:159-170.

Hatton GI (2002) Glial-neuronal interactions in the mammalian brain. Adv Physiol Educ 26:225-237.

Haydon PG (2000) Neuroglial networks: Neurons and glia talk to each other. Curr Biol 10:R712-714.

Haydon PG (2001) Glia: listening and talking to the synapse. Trends Neurosci 2:185-193.

Hegstad E, Langmoen IA, Hablitz JJ (1989) Zinc and glycine do not modify low-magnesium-induced epileptiform activity in the immature neocortex in vitro. Epilepsy Res 3:174-177.

Ichikawa M, Muramoto K, Kobayashi K, Kawahara M, Kuroda Y (1993) Formation and maturation of synapses in primary cultures of rat cerebral cortical cells: an electron microscopic study. Neurosci Res 16:95-103.

Kandler K, Katz LC (1995) Neuronal coupling and uncoupling in the developing nervous system. Curr Opin Neurobiol 5:95-105.

Khazipov R, Holmes GL (2003) Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo. J Neurosci 23:5337-5341.

Lin Z, Geng L, Li R, He X, Zheng JQ, Xie Z (2003) Frequency modulation of synchronized Ca2+ spikes in cultured hippocampal networks through G-protein-coupled receptors. J Neurosci 23:4156-4163.

Long MA, Cruikshank SJ, Jutras MJ, Connors BW (2005) Abrupt Maturation of a Spike-Synchronizing Mechanism in Neocortex. J Neurosci 25:7309-7316.

Luhmann HJ, Prince DA (1991) Postnatal maturation of the GABAergic system in rat neocortex. J Neurophysiol 65:247-263.

Miles R, Wong RKS (1983) Single neurones can initiate synchronized population discharged in the hippocampus. Nature 306:307-373

Mirollo RE, Strogatz SH (1990) Synchronization of pulse-coupled biological oscillations. SIAM J Appl Math 50:1645-1662.

Muramoto K, Ichikawa M, Kawahara M, Kobayashi K, Kuroda Y (1993) Frequency of synchronous oscillations of neuronal activity increases during development and is correlated to the number of synapses in cultured cortical neuron networks. Neurosci Lett 163:163-165.

Murphy TH, Blatter LA, Wier WG, Baraban JM (1992) Spontaneous synchronous synaptic calcium transients in cultured cortical neurons. J Neurosci 12:4834-4845.

Nakanishi K, Okouchi Y, Ueki T, Asai K, Isobe I, Eksioglu YZ, Kato T, Hasegawa Y, Kuroda Y (1994) Astrocytic contribution to functioning synapse formation estimated by spontaneous neuronal intracellular Ca2+ oscillations. Brain Res 659:169-178.

Nedergaard M (1994) Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science 263:1768-1771.

Nett WJ, Oloff NSH, McCarthy KD (2002) Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity. J Neurosci 87:528-537.

Ogura A, Iijima T, Amano T, Kudo Y (1987) Optical monitoring of excitatory synaptic activity between cultured hippocampal neurons by a multi-site Ca2+ fluometry. Neurosci Lett 78:69-74.

Parpura V, Haydon PG (1999) UV photolysis using a micromanipulated optical fiber to deliver UV energy directly to the sample. J Neurosci Meth 87:25-34.

Pfrieger FW, Barres BA (1996) New views of synapse-glia interactions. Curr Opin Neurobiol 6:615-621.

Pikovsky A, Rosenblum M, Kurths J (2001) Synchronization: A universal concept in nonlinear sciences. Cambridge University Press, Cambridge, U.K.

Robinson HPC, Kawahara M, Jimbo Y, Torimitsu K, Kuroda Y, Kawana A (1993) Periodic synchronized bursting and intracellular calcium transients elicited by low magnesium in cultured cortical neurons. J Neurophysol 70:1606-1616.

Rogawski MA (2005) Astrocytes get in the act in epilepsy. Nat Med 11:919-920.

Strogatz SH (2001) Exploring complex networks. Nature 410:268-276.

Taylor CP, Dudek FE (1982) Synchronous neural afterdischarges in rat hippocampal slices without active chemical synapses. Science 218:810-812.

Telfeion AE, Connors BW (1999) Epileptiform propagation patterns mediated by NMDA and non-NMDA receptors in reat neocortex. Epilepsia 40:1499-1506.
Tian GF, Azmi H, Takano T, Xu Q, Peng W, Lin J, Oberheim NA, Lou N, Wang X, Zielke HR, Kang J, Nedergaard M (2005) An astrocytic basis of epilepsy. Nat Medicine 11:973-981.

Traub RD, Wong RKS (1982) Cellular mechanism of neuronal synchronization in epilepsy. Science 216:745-747.

Traub RD, Bibbig A, LeBeau FE, Cunningham MO, Whittington MA (2005) Persistent gamma oscillations in superficial layers of rat auditory neocortex: experiment and model. J Physiol (Lond) 562:3-8.

Uiilian EM, Sapperstein SK, Christopherson KS, Barres BA (2001) Control of synapse number by glia. Science 291:657-661.

Wang XS, Gruenstein EI (1997) Mechanism of synchronized Ca2+ oscillations in cortical neurons. Brain Res 767:239-249.

Kamioka H, Maeda E, Jimbo Y, Robinson HPC, Kawana A (1996) Spontaneous periodic synchronized bursting during formation of mature patterns of connections in cortical cultures. Neuronsci Lett 206:109-112.

Wong ROL, Meister M, Shatz CJ (1993) Transient period of correlated bursting activity during development of the mammalian retina. Neuron 11:923-938

Yuste R, Peinado A, Katz LC (1992) Neuronal domains in developing neocortex. Science 257:665-669.
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