Synapses are continuously formed and eliminated throughout life in the mammalian brain, and emerging evidence suggests that this structural plasticity underlies experience-dependent changes of brain functions such as learning and long-term memory formation. However, it is generally difficult to understand how the rewiring of synaptic circuitry observed in vivo eventually relates to changes in animal's behavior. This is because afferent/efferent connections and local synaptic circuitries are very complicated in most brain regions, hence it is largely unclear how sensorimotor information is conveyed, integrated, and processed through a brain region that is imaged. The cerebellar cortex provides a particularly useful model to challenge this problem because of its simple and welldefined synaptic circuitry. However, owing to the technical difficulty of chronic in vivo imaging in the cerebellum, it remains unclear how cerebellar neurons dynamically change their structures over a long period of time. Here, we showed that the commonly used method for neocortical in vivo imaging was not ideal for long-term imaging of cerebellar neurons, but simple optimization of the procedure significantly improved the success rate and the maximum time window of chronic imaging. The optimized method can be used in both neonatal and adult mice and allows time-lapse imaging of cerebellar neurons for more than 5 mo in ϳ80% of animals. This method allows vital observation of dynamic cellular processes such as developmental refinement of synaptic circuitry as well as long-term changes of neuronal structures in adult cerebellum under longitudinal behavioral manipulations.long-term, time-lapse imaging; two-photon microscopy; development; plasticity IN VIVO TIME-LAPSE MICROSCOPY is now extensively used for imaging fine neuronal structures and activity in the brain of living animals. These experiments have revealed the dynamic nature of synaptic wiring and functional architecture of neuronal ensembles, providing novel insights into experience-dependent changes of brain functions (Bhatt et al. 2009;Chen and Nedivi 2010;Huber et al. 2012). However, it is generally difficult to understand how the formation and elimination of synapses modify the function of local brain circuitry, and eventually, an animal's behavior.The cerebellum provides a unique advantage to challenge this problem. Unlike most other brain regions, extensively characterized anatomical and physiological properties of cerebellar neurons provide testable models regarding how sensorimotor information is conveyed and integrated, and how plas-

Crousillac S, Colonna J, McMains E, Dewey JS, Gleason E. Sphingosine-1-phosphate elicits receptor-dependent calcium signaling in retinal amacrine cells. J Neurophysiol 102: 3295-3309, 2009. First published September 23, 2009 doi:10.1152/jn.00119.2009. Evidence is emerging indicating that sphingosine-1-phosphate (S1P) participates in signaling in the retina. To determine whether S1P might be involved in signaling in the inner retina specifically, we examine the effects of this sphingolipid on cultured retinal amacrine cells. Whole cell voltage-clamp recordings reveal that S1P activates a cation current that is dependent on signaling through G i and phospholipase C. These observations are consistent with the involvement of members of the S1P receptor family of G-proteincoupled receptors in the production of the current. Immunocytochemistry and PCR amplification provide evidence for the expression of S1P1R and S1P3R in amacrine cells. The receptor-mediated channel activity is shown to be highly sensitive to blockade by lanthanides consistent with the behavior of transient receptor potential canonical (TRPC) channels. PCR products amplified from amacrine cells reveal that TRPCs 1 and 3-7 channel subunits have the potential to be expressed. Because TRPC channels provide a Ca 2ϩ entry pathway, we asked whether S1P caused cytosolic Ca 2ϩ elevations in amacrine cells. We show that S1P-dependent Ca 2ϩ elevations do occur in these cells and that they might be mediated by S1P1R and S1P3R. The Ca 2ϩ elevations are partially due to release from internal stores, but the largest contribution is from influx across the plasma membrane. The effect of inhibition of sphingosine kinase suggests that the production of cytosolic S1P underlies the sustained nature of the Ca 2ϩ elevations. Elucidation of the downstream effects of these signals will provide clues to the role of S1P in regulating inner retinal function.

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