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

Abstract

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.

Figure 1.

In vivo two-photon images of EGFP-labeled neurons in the cerebellar cortex of an adult mouse. Neurons were labeled with a cortical injection of AAV-EGFP and subsequently imaged in vivo through a cranial window. Maximum projections of parallel fibers (A), Purkinje cell (B), molecular layer interneuron (C), and granule cells (D) are shown. B, The image was digitally rotated to yield a sagittal view of the Purkinje cell. D, Mediolateral (M–L) and rostrocaudal (R–C) axes of the images. Arrowheads indicate claw-shaped endings of granule cell dendrites.

Figure 2.

A small fraction of PF varicosities appears or disappears. Exemplar time-lapse images of PFs and their varicosities in two different preparations (PF1 and PF2) are shown. Overall imaging periods were 45 and 2 weeks (w) for PF1 and PF2, respectively. Arrows indicate PF varicosities that appeared in a particular imaging session. Arrowheads indicate PF varicosities that disappeared in the next imaging session. Neither an arrow nor an arrowhead is drawn in the image taken at 45 weeks because there were multiple imaging sessions between the 4 week and 45 week time points (images not shown). The histogram shows the fraction of dynamic varicosities. Only PFs that have ≥3 time points (0, 2, and 4 weeks) were analyzed and fractions of appeared and disappeared varicosities between two consecutive time points (0–2 w, 2–4 w) were quantified. The fractions represented by the right bar (0–4 w) indicate the total number of appeared and disappeared varicosities divided by total number of varicosities over this 4 week period.

Figure 3.

Mice are able to perform acrobatic motor skill learning task regardless of the presence of a cranial window and the occasional use of anesthesia for time-lapse in vivo imaging. A, Mice trained on the acrobatic task. The line graphs represent number of error (B,D) and latency (C,E) to traverse an elevated course for acrobatic motor skill learning during the 40 d of training period. B, C, Individual mice. D, E, Average of all mice. Error bars represent SE of the mean.

Figure 4.

Acrobatic motor skill learning suppresses dynamic structural plasticity of PF varicosities. A, Timeline of the experiment. B, Exemplar time-lapse images of PFs and their varicosities before, during, and after the training of acrobatic motor skill learning. Arrowheads indicate PF varicosities that disappeared in the next imaging session. C, The histogram represents the fraction of dynamic varicosities between two consecutive time points shown in A over the 12 week imaging period under the acrobatic motor skill learning task. Inset, A fraction of dynamic varicosities during the initial 10 d of training. These time points are available only in a subset of PFs. D, The histogram represents the fraction of dynamic varicosities between two consecutive time points over the 9 week imaging period under the motor activity-matched control task. The fractions shown on the right bar (2–5 Total) indicate the total number of appeared and disappeared varicosities divided by the total number of varicosities over this 7 week period.

Figure 5.

Acrobatic motor skill learning primarily suppresses the formation of new varicosities. A, The histogram represents the average size of stable, preexisting-disappeared, and newly appeared varicosities in the motor activity-matched control group (MO) and the acrobatic training group (AC). Error bars represent SD. *p < 0.05. B, The histogram shows the fraction of stable, preexisting-disappeared, and newly appeared varicosities in the MO and the AC groups. **p < 0.01. C, Exemplar time-lapse images of a newly appeared varicosity that disappeared by the next imaging session. The images were taken from the MO group. Arrows indicate the location of the varicosity.