Long-term in vivo time-lapse imaging of synapse development and plasticity in the cerebellum

Abstract

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-

Chronic imaging of movement-related Purkinje cell calcium activity in awake behaving mice

“…This method produced sufficient pressure on the brain to reduce motion artifacts without causing clear signs of damage. In contrast, we found that additional pressure created by placing the coverslip inside the skull opening (Nishiyama et al 2014) or by putting agarose between the coverslip and brain (Hoogland et al 2015) caused damage to some PCs that became apparent over time (data not shown).…”

“…These cerebellar regions are related to sensorimotor functions of the orofacial area including the tongue (Bryant et al , 2010Welsh et al 1995). Chronic imaging requires a stable, clear cranial window, a technique reported to be difficult in the mouse cerebellum (Nishiyama et al 2014). We found that three factors could help improve our success rate.…”

“…The inability to maintain a clear window long enough to image, particularly after viral injections, could account for this scarcity. PCs, with elaborate dendritic arbors approaching the pial surface, may be highly amenable to damage during surgery, and problems creating a long-lasting window over the cerebellum (Nishiyama et al 2014) contribute to this difficulty. Here we installed a stable cranial window over healthy cerebellar lobules crus I and II and tested GECIs expressed specifically in PCs.…”

Membrane Trafficking in Neuronal Development: Ins and Outs of Neural Connectivity

“…Dendritic spines are typically mushroom-shaped dendritic projections that are malleable, changing size and shape in response to activity and external cues. Such synaptic plasticity continues into adulthood and is associated with learning and memory (Chen et al, 2012; Hamilton et al, 2012; Holtmaat and Svoboda, 2009; Nishiyama et al, 2014; Trachtenberg et al, 2002). …”

Neuregulin1 displayed on motor axons regulates terminal Schwann cell-mediated synapse elimination at developing neuromuscular junctions

“…This developmental transformation includes elimination and remodeling of existing synapses, commonly termed "synapse elimination," and is seen throughout the nervous system (1,2). Similar remodeling can occur throughout life during learning, aging, and disease, however (3)(4)(5).…”