Role of Synchronous Activation of Cerebellar Purkinje Cell Ensembles in Multi-joint Movement Control - PubMed (original) (raw)

Role of Synchronous Activation of Cerebellar Purkinje Cell Ensembles in Multi-joint Movement Control

Tycho M Hoogland et al. Curr Biol. 2015.

Abstract

It is a longstanding question in neuroscience how elaborate multi-joint movements are coordinated coherently. Microzones of cerebellar Purkinje cells (PCs) are thought to mediate this coordination by controlling the timing of particular motor domains. However, it remains to be elucidated to what extent motor coordination deficits can be correlated with abnormalities in coherent activity within these microzones and to what extent artificially evoked synchronous activity within PC ensembles can elicit multi-joint motor behavior. To study PC ensemble correlates of limb, trunk, and tail movements, we developed a transparent disk treadmill that allows quantitative readout of locomotion and posture parameters in head-fixed mice and simultaneous cellular-resolution imaging and/or optogenetic manipulation. We show that PC ensembles in the ataxic and dystonic mouse mutant tottering have a reduced level of complex spike co-activation, which is delayed relative to movement onset and co-occurs with prolonged swing duration and reduced phase coupling of limb movements as well as with enlarged deflections of body-axis and tail movements. Using optogenetics to increase simple spike rate in PC ensembles, we find that preferred locomotion and posture patterns can be elicited or perturbed depending on the behavioral state. At rest, preferred sequences of limb movements can be elicited, whereas during locomotion, preferred gait-inhibition patterns are evoked. Our findings indicate that synchronous activation of PC ensembles can facilitate initiation and coordination of limb and trunk movements, presumably by tuning downstream systems involved in the execution of behavioral patterns.

Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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Figures

Figure 1

Figure 1

A Transparent Disk Treadmill (A) Mice with head posts are mounted in a head post holder that is attached to a central bridge overlaying a rotary transparent Plexiglas disk on which they can walk at will due to low-friction ball bearings and low disk inertia. A mirror placed underneath the disk reflects an image of the mouse as seen from below onto an infrared-sensitive CCD camera. The mouse is illuminated by two strips of LEDs (940 nm), with one mounted on the mirror and the other directly opposite, both at 45° angles. An optical filter placed between the mirror and camera blocks light from two-photon laser excitation during imaging sessions or optogenetic stimulation while passing light used for illumination of the mouse. A magnetic encoder centered above the disk in conjunction with a magnet placed in the center of the rotary disk allows recording of disk position as a voltage signal. A computer-controlled electromagnetic clutch placed along the rotational axis can be engaged for brief durations to generate high saliency sensory perturbations. (B) Image of a mouse as captured by the CCD camera (disk edge indicated in white). (C) Top: frames taken during different phases of mouse locomotion. Paws are indicated by the colored dots overlaid on each frame, while body axis (b.a.) and tail axis (t.a.) are indicated by the white and red dashed lines, respectively. Bottom: x displacement of the four paws of a mouse are represented in colors corresponding to the identified paw objects in the top panels with directly below the state vectors representing paw stance and swing periods. Vertical gray lines denote the time points during locomotion that correspond to the frames shown in the top panels. LF, left front limb; RF, right front limb; LH, left hind limb; RHL, right hind limb. See also Figure S1.

Figure 2

Figure 2

Motor Deficits in tg/tg Mice Analyzed with a Disk Treadmill (A) Left: body (b.a.) and tail (t.a.) axis and paw positions (colored dots) extracted from a tg/tg mice walking on the transparent disk treadmill. The white arrow denotes a misstep. Right: paw displacements (in x) and corresponding gait patterns in a tg/tg mouse. (B) Cumulative frequency histograms of treadmill speed (top) and running bout duration (bottom) in wild-type and tg/tg mice. Both were not significantly different (speed, p = 0.7; bout duration, p = 0.29). (C) Stance and swing times for wild-type and tg/tg mice (mean ± SEM). (D) Coupling between limbs shows deficits in tg/tg mice. Examples are shown of the increased out-of-phase placement of limbs in tg/tg relative to wild-type mice. (E) Body and tail angles showed significantly larger deflections in tg/tg than in wild-type mice. See also Figure S2.

Figure 3

Figure 3

Altered Timing and Reduced Levels of Locomotion-Associated Microzonal CS Co-activation in tg/tg Mice (A) Two-photon optical sections of the cerebellar cortex bolus-loaded with OGB-1/AM with superimposed PC dendrite masks extracted with spatial independent component analysis (scale bar, 10 μm). Traces from the bright dendrite (pink) and extracted CS events (gray triangles) are shown for both wild-type and tg/tg mice together with disk encoder speed and the number of PC dendrites with co-active CS events. (B) Synchronous CS events as triggered by locomotion onset. Co-active events were binned with ms precision using a hyperacuity algorithm. In tg/tg mice, peak event synchrony was delayed relative to wild-types with respect to locomotion onset. When CS co-active events were triggered off of sensory perturbations evoked with the magnetic clutch of the disk treadmill (red lines), elicited twitches resulted in similarly timed CS synchrony. (C) Spatial correlations of CS events in wild-type mice showed increased correlation during locomotion. Such correlation increases were not observed in tg/tg mice during locomotion. Shaded regions indicate the SEM. See also Figure S3.

Figure 4

Figure 4

State-Dependent Motor Behavior during Selective Optogenetic Activation of PCs (A) Left: optical section of the molecular layer of the cerebellum acquired with a two-photon microscope indicating the presence of the ChR2(H134)-EYFP fusion protein. Right: closeup of the boxed region shows expression of ChR2(H134)-EYFP in sagitally arranged PC dendrites (the outline denotes a single arbor; a, anterior; p, posterior). (B) Schematic indicating the region of optogenetic stimulation (lobule V). Mice were mounted on the treadmill, and a 400 μm fiber was mounted above a small craniotomy above the medial cerebellar vermis lobule V for PC stimulation. A blue LED acted as light source and was coupled into the fiber giving a mean output of 1–3 mW at the fiber tip. (C) Optogenetic stimulation of PCs (250 ms, 465 nm; shaded blue areas) in behaving mice resulted in stereotyped behaviors that were state dependent. Stimulation during rest could evoke twitches (see also Figures S3A and S3B) or initiate stepping behavior, whereas stimulation during the step cycle could slow down or stop locomotion. (D) Treadmill disk speeds (normalized, individual traces, gray; average response, black) showing halting (top) or initiation (bottom) of locomotion when PCs are optogenetically stimulated. (E) Camera frame differences at three time intervals following optogenetic stimulation offset demonstrating that PC stimulation during rest triggered stepping on the disk treadmill. (F) The four most common stepping patterns are depicted in red and illustrate the placement order of each paw for a sequence of four steps. (G) Optogenetically induced and spontaneous walk patterns were comparable in that most frequently a front paw was first lifted followed by a diagonally apposed hind limb. (H) Disk speeds during slowdown of locomotion (individual traces, gray; average response, black) compared to average response during halting of locomotion (red). Speeds did not differ significantly prior to slowdown or halting of locomotion. (I) Speed during spontaneous walking prior to the stimulus onset does not determine the timing of when the animal comes to a complete halt. (J) PC stimulation during locomotion causes interruption of the step cycle. Shown are trials of normalized paw displacements (in x) for three mice triggered off of the onset of a 250 ms light stimulus. See also Movies S1, S2, and S3.

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