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

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 well-defined 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 ∼80% 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.

The long-term structural plasticity of cerebellar parallel fiber axons and its modulation by motor learning.

Presynaptic axonal varicosities, like postsynaptic spines, are dynamically added and eliminated even in mature neuronal circuitry. To study the role of this axonal structural plasticity in behavioral learning, we performed two-photon in vivo imaging of cerebellar parallel fibers (PFs) in adult mice. PFs make excitatory synapses on Purkinje cells (PCs) in the cerebellar cortex, and long-term potentiation and depression at PF-PC synapses are thought to play crucial roles in cerebellar-dependent learning. Time-lapse vital imaging of PFs revealed that, under a control condition (no behavioral training), ∼10% of PF varicosities appeared and disappeared over a period of 2 weeks without changing the total number of varicosities. The fraction of dynamic PF varicosities significantly diminished during training on an acrobatic motor skill learning task, largely because of reduced addition of new varicosities. Thus, this form of motor learning was associated with greater structural stability of PFs and a slight decrease in the total number of varicosities. Together with prior findings that the number of PF-PC synapses increases during similar training, our results suggest that acrobatic motor skill learning involves a reduction of some PF inputs and a strengthening of others, probably via the conversion of some preexisting PF varicosities into multisynaptic terminals.

Dendritic translocation establishes the winner in cerebellar climbing fiber synapse elimination.

In many regions of the developing mammalian nervous system, functional synaptic circuitry is formed by competitive elimination of early formed redundant synapses. However, how winning synapses emerge through competition remains unclear in the brain largely because of the technical difficulty of directly observing this dynamic cellular process in vivo. Here, we developed a method of two-photon multicolor vital imaging to observe competitive elimination of supernumerary climbing fibers (CFs) in the cerebellum of live mouse pups. At birth, each Purkinje cell (PC) in the cerebellar cortex is innervated by multiple CFs; an activity-dependent regression of supernumerary CFs ultimately yields a single innervation for most PCs by postnatal day 21. As supernumerary CFs are pruned, the terminal field of CFs translocates from the soma to the dendrites of PCs. In vivo time-lapse imaging of CF elimination revealed that (1) CF terminals were highly motile on the soma, but their motility was significantly reduced on dendrites; (2) only one CF could translocate to the dendrites whereas their competitors were restricted to perisomatic regions; and (3) the CF that began dendritic translocation became the winner. Moreover, selective photo-ablation of the winning CF (that undergoes dendritic translocation) reversed the fate of its losing competitor. These results indicate that dendritic translocation is a key cellular event that determines the winner during CF elimination. We propose that CF terminals are selectively stabilized on dendrites, providing irreversible competitive vigor to the first CF to form dendritic synapses.

A single exposure to voluntary ethanol self-administration produces adaptations in ethanol consumption and accumbal dopamine signaling.

In well-trained animals, accumbal dopamine release is stimulated during operant ethanol self-administration, but the time course of development of this dopaminergic response, particularly during the acquisition of ethanol drinking behavior, remains unknown. To examine this, we trained male Long-Evans rats to self-administer 10% ethanol plus 10% sucrose, using a protocol in which the concentration of ethanol was kept constant throughout the study. The animals were required to press the lever four times to gain continuous access to the drinking solution for 20 minutes, and microdialysis was performed on either the first or second day of 10% ethanol plus 10% sucrose self-administration or 10% sucrose as controls. Ethanol and dopamine were both analyzed in the dialysates. All groups (day 1 and 2 ethanol and their corresponding sucrose controls) showed an increase in accumbal dopamine during the transfer from the home cage into the operant chamber. Our main finding was an increase in dopamine in the nucleus accumbens core-shell border during the first 5 minutes of consumption on the second day but not on the first day of ethanol self-administration. Our results suggest that a single exposure to a 10% ethanol plus 10% sucrose drinking solution may be sufficient to learn the association between ethanol cues and its reinforcing properties. Furthermore, we speculate that the dopamine response during ethanol consumption likely reflects the reward-prediction role of the mesolimbic dopamine system.

A 3-day exposure to 10% ethanol with 10% sucrose successfully initiates ethanol self-administration.

The initiation phase of ethanol self-administration is difficult to study using the well-established, sucrose-fading procedure due to the changing concentrations of ethanol in the first few days. The purpose of this experiment was to test whether a modified sucrose-substitution procedure in which rats are initially exposed to high concentrations of ethanol and sucrose for three days would successfully initiate ethanol self-administration. Male Long-Evans rats were trained to lever-press with a 10% sucrose solution in which four or 20 responses allowed 20-min access to the solution. Subsequently, rats were exposed to a 3-day period of operant self-administration of 10% sucrose+10% ethanol. This constant-concentration exposure was followed by the standard procedure in which sucrose is completely faded out. The establishment of ethanol self-administration was determined by ethanol intake, pre- and postprocedure two-bottle choice preference tests, and extinction trials. The mean ethanol intake was 2.2 times higher on day 2 compared with day 1 on the 10% sucrose+10% ethanol solution. After fading out the sucrose, the daily intake of 10% ethanol solution over 5 days was stable at approximately 0.57 g/kg. Ethanol preference was approximately threefold higher after the modified sucrose-fading procedure. Responding during a single session extinction test was dramatically increased from 4 to 61+/-13 or 20 to 112+/-22 responses in 20 min. Similar to the standard sucrose-fading method, we did not observe a significant relationship between extinction responding and ethanol intake. Blood alcohol concentrations were 4.5 mM 20 min after consumption began. We conclude that initiation and establishment of ethanol self-administration will occur using this modified sucrose-fading procedure.