| Abstract No.: | C-B3035 |
| Country: | Canada |
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| Title: | CA2+ DEPENDENT GLUTAMATE RELEASE FROM ASTROCYTES: A MECHANISM FOR RECRUITING GROWTH CONES TO REGIONS OF ACTIVITY. |
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| Authors/Affiliations: | 1 Johanna Hung; 1 Michael Colicos*;
1 University of Calgary, AB, Canada
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| Content: | Objectives: To investigate activity driven changes in neuronal connectivity
Materials and Methods: This study is based on an in vitro dissociated rat hippocampal-astrocyte coculture paradigm, in which growth cone dynamics are observed during network development. A non-invasive photoconductive stimulation protocol was used to depolarize astrocytes 42µm and 126 µm away from one side of axonal growth cones (1). Live cell imaging was used to monitor growth cone behaviour in neurons transfected with fluorescent proteins. A pharmacological approach was taken to investigate the biochemical mechanism of the observed phenomena.
Results: Long protrusions of actin rich growth cone filopodia extended towards regions of induced neuronal activity. Filopodial extension was preceded by the relocation of GFP-VAMP-2 to regions of the membrane where filopodial extension later occurred, supporting the hypothesis that SNARE proteins are involved in membrane expansion at the tip of growing neurites (2). Long term turning studies indicated that altered filopodial arrangement results in long-lasting change in growth cone orientation, turning toward the source of the activity. Importantly, the attraction was abolished for stimulations at 126µm but not 42µm when the connexion 43 blocker carbenoxolone (50µM) was added. In this culture/stimulation paradigm, glial depolarization and Ca2+ wave propogation occurs, either as a result of neuronal activity or by direct depolarization. Thus, blocking the effect with CBX suggests glial Ca2+ wave transmission is critical for this axonal guidance mechanism. This attractive effect was calcium dependent and blocked by CNQX (10µM), a non-NMDA glutamate receptor antagonist.
Conclusion: Here we show evidence for a novel mechanism of activity driven axon guidance that involves the communication between neuronal growth cones and the astrocytes that they are in contact with. We propose a model whereby Ca2+ dependent release of glutamate from the astrocytes directs growth cone dynamics in a spatially and temporally defined way. This establishes localized brain activity is an attractive cue for axonal growth cone guidance. This result is interesting with respect to the observation that direct, exogenous application of glutamate gradients trigger repulsive responses in both axonal and dendritic growth cones. In our study glutamate may be released by underlying astrocytes, and in such allows for the attractive turning effect. We propose this activity driven axonal guidance mechanism as a critical part of not only the development of neuronal connectivity, but as a mechanism for structural plasticity and regenspeculate that the response of growth cones to glutamate is regulated very tightly and depends highly on the concentration of the cue, and possibly on additional cues coreleased with glutamate from astrocytes during depolarization.
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