Locomotion is a unique yet fundamental behavior that enables animals to move in space and survive in various

environmental conditions. To maintain a stable and coordinated gait when surrounding conditions change, animals
adjust appropriately their “default” motor patterns via locomotion adaptation. Locomotion adaptation is, a form of
motor learning occurring through trial and error in a short timescale (seconds to minutes). Changes in the limbs
coordination during locomotion were extensively investigated using split-belt treadmills, allowing to introduce
different speeds for each side of the body, forcing subjects to adapt their interlimb parameters, such as step
length, to restore movement symmetry.

It is believed that Llocomotion adaptation is dependent on the cerebellum, which is considered to be an important
brain structure for coordination of limbs and processing of sensorimotor errors. to perform smooth and calibrated
movements. Cerebellar damage often(?) results in uncoordinated gait, or ataxia - people with this pathology
experience difficulties with the speed adaptation when walking on a split-belt treadmill and are , unable to restore
movement symmetry. Besides cerebellar damage, aAtaxia is observed in several motor and non-motor disorders,
for instance spinocerebellar ataxias, Huntington’s disease and autism. So, understanding of what cerebellar
mechanisms drive limbs adaptation could provide valuable insights for developing symptomatic treatment and
rehabilitative techniques for such diseases.

However, how cerebellum is involved in the adaptive changes in the interlimb coordination even during walking is
still unclear.

What we know, dDuring locomotion, information about environmental changes and kinematic parameters of
movements is transmitted to the Purkinje cells' (PC) dendrites in cerebellar cortex via a system of parallel and
climbing fibers, triggering PC to generate simple and complex spikes (CS) respectively. It is known that CS events
during walking are moderately tuned to the step phase, however they cannot encode the response to the external
stimuli or stimuli per se given their low frequency firing. So, there should be different mechanisms of encoding
such information during locomotion, for instance, via increased amplitude rate or enhanced synchronous response
from several PCs.

The CS activity during locomotion was has been widely studied, however the involvement of CF inputs to the
coordination of limbs remains largely unknown.

Some experiments have shown that perturbation of a single limb in locomoting animals resulted in increased
probability of CS firing and significantly enhanced synchronous activation of CF inputs in many PCs. These changes
were observed mostly in a swing phase of stepping, suggesting the possible preparation of a limb for timely
placement on a treadmill. However, there is no evidence of how CF inputs may regulate the adaptive changes in
the coordination between several limbs.

I speculate that if the CS activity is modulated with the speed of movement and reflects the perturbation
occurrence to the single limb, then the speed increase to the one side of the animal’s body could result in
analogous modulation of CF inputs determining the gait change.

My hypothesis therefore is that dendritic CS activity encode adaptive adjustments in the coordination of ipsilateral
limbs of a locomoting mouse.

So, in my project, I will investigate for the first time the role of DCS activity of PC during locomotion adaptation in
mice.
To achieve this goal, I will first address whether perturbation of the one side of the body results in changed
interlimb and intralimb coordination. I will build a special split-belt treadmill with the possibility to fix mice and
have bottom view of limbs movements. I will apply the same speed for the whole-body movement as the baseline
period and

then will increase the speed for one side of the body. I will use DeepLabCut software for labeling the limbs
positions on the behavioral movies and further will calculate the step length and stride length based on the labeled
points.

To visualize activity of Purkinje cells dendrites, I will first perform chronic cranial window surgery above vermal and
paravermal area of cerebellum in L7-Cre mice with subsequent injection of adeno-associated viral vector, to
express specifically in PCs GCaMP6f for calcium imaging. By uUsing two-photon calcium imaging, I will be able to
image CF-evoked calcium events in the PC dendrites, characterizing dendritic CS.

Next, I will perform experiments on the imaging of the dendritic CS simultaneously with the recording of locomotor
behavior before and after speed change. To illustrate putative changes in the amplitude of DCS in single PCs, I will
compute the fluorescence traces dF/F in each dendrite to identify CS-associated calcium transients and then to
analyze difference between values before and after perturbation.

To demonstrate possible encoding of a speed change by the increased coherence and rate in DCS firing in multiple
PCs, I will calculate the co-activation index of the PC dendrites before and after the perturbation.

In addition, I will address whether changes in DCS firing are related to the steps adjustments during adaptation, by
combining prior analysis of DCS activity and behavioral parameters, such as step length and stride length.

As for my progress, besides learning basicmastering the methods used in our my lab, such as cranial surgery and
viral injections, I managed to build the special split-belt treadmill with the head-fixation system. This setup consists
of two transparent wheels made of acrylic plastic and that are connected to the motors, allowing which allow for
independently wheel rotation. The head-fixation bars keep the head immobile during neuronal activity. A mirror
under 45 degrees between wheels allows to have side and bottom view, recorded by the camera attached to the
setup.

In my pilot experiments I developed a protocol for head-fixed mice walking on such motorized treadmill with the
same speed for each side of the body. Next, I worked out a behavioral training for the split condition, when the
speed for one side of the body changes. In my preliminary results I found that head-fixed locomoting mice are
more willing to adapt to the faster speed, than slower. Additionally, for both types of trainings, I observed that
front limbs are better adjusting to the speed change then the hind limbs, which is consistent with the prior
behavioral studies of locomotion adaptation.

My next steps would be to perform viral injections to the cerebellar PCs layer, imaging of PCs dendrites with two-
photon Ca imaging and subsequent analysis of dendritic CS and changes in limbs coordination.

To sum up.

Investigation of a role of CS in the interlimb coordination during locomotion may provide valuable insights to the
development of new therapeutic targets for the disorders characterized by uncoordinated gait.

Also, uUnderstanding about the cerebellar mechanisms underlying the fine motor control of complex movements
could contribute not only to the medicine, but also to the robotic engineering, where programs controlling limbs
movements in terrestrial robots, are developed based on the computations happening in the animal’s cerebellum.
Therefore, I think my work will might reveal the previously undiscovered role of CS activity during locomotion
adaptation in mice and will provide important knowledge to the understanding of cerebellar control of
movements.