Takayuki Michikawa

The coordination of various brain regions achieves both volitional and forced motor control, but the role of the primary motor cortex in proficient running motor control remains unclear. This study trained mice to run at high performance (>10,000 rotations per day or >2,700 rotations per hour) using a running wheel, and then assessed the effects of the removal of bilateral cortical areas including the primary motor cortex on volitional (self-initiated) and forced (externally driven) running locomotion. The control sham-operated group revealed a quick recovery of volitional running, reaching half of the maximum daily rotation in 3.9 ± 2.6 days (n = 10). In contrast, the cortical injury group took a significantly longer period (7.0 ± 3.3 days, n = 15, p < 0.05) to reach half of the maximum volitional daily rotation, but recovered to preoperative levels in about two weeks. Furthermore, even 3 days after surgery to remove cortical regions, the running time on a treadmill moving at 35.3 cm/s, which is difficult for naïve mice to run on, was not significantly different from that in the sham-operated group. These results suggest that the intact primary motor cortex is not necessarily required to execute trained fast-running locomotion, but rather contributes to the spontaneity of running in mice.

Optogenetics^{1 – 3} is a powerful tool for elucidating the link between neuronal circuit activity and animal behavior. Efficiently accomplishing this elucidation requires precise control of the firing timing of multiple target cells arranged in three dimensions by photostimulation with single-cell resolution while monitoring neuronal circuit activity. The two-photon (2P) excitation method^4,5 with spatially tight focusing at a high numerical aperture (NA) can localize the photostimulated region to a single-cell size in the axial direction. However, activation by spatially tight focusing requires lateral scanning of the laser over a single cell. The illumination spot can be expanded to single-cell size by computer-generated holography (CGH);⁶ however, as with low-NA focusing, the axial excitation region increases in proportion to the spot size.⁷ In addition, the quality of the excitation beam worsens because of speckle noise resulting from the interference effect of phase modulation. Thus, the trade-off between temporal and axial resolution has become a significant obstacle.

The coordination of various brain regions achieves both volitional and forced motor control, but the role of the primary motor cortex in proficient running motor control remains unclear. This study trained mice to run at high performance (>10,000 rotations per day or >2,700 rotations per hour) using a running wheel, and then assessed the effects of the removal of bilateral cortical areas including the primary motor cortex on volitional and forced running locomotion. The control sham-operated group revealed a quick recovery of volitional running, reaching half of the maximum daily rotation in 3.9 +/-2.6 days (n = 10). In contrast, the cortical injury group took significantly a longer period (7.0 +/-3.3 days, n = 15) to reach half of the maximum volitional daily rotation, but recovered to preoperative levels in about two weeks. Furthermore, even 3 days after surgery to remove cortical regions, the running time on a treadmill moving at 35.3 cm/sec, which is difficult for naïve mice to run on, was not significantly different from that in the sham-operated group. These results suggest that the intact primary motor cortex is not necessarily required to execute trained fast-running locomotion, but rather contributes to the spontaneity of running in mice.

Background: In the cerebellar cortex, Purkinje cells are the only output neurons and exhibit two types of discharge. Most Purkinje cell discharges are simple spikes, which are commonly appearing action potentials exhibiting a rich variety of firing patterns with a rate of up to 400 Hz. More infrequent discharges are complex spikes, which consist of a short burst of impulses accompanied by a massive increase in dendritic Ca ²⁺ with a firing rate of around 1 Hz. The discrimination of these spikes in extracellular single-unit recordings is not always straightforward, as their waveforms vary depending on recording conditions and intrinsic fluctuations. New Method: To discriminate complex spikes from simple spikes in the extracellular single-unit data, we developed a semiautomatic spike-sorting method based on divisive hierarchical clustering. Results: Quantitative evaluation using parallel in vivo two-photon Ca ²⁺ imaging of Purkinje cell dendrites indicated that 96.6% of the complex spikes were detected using our spike-sorting method from extracellular single-unit recordings obtained from anesthetized mice. Comparison with Existing Method(s): No reports have conducted a quantitative evaluation of spike-sorting algorithms used for the classification of extracellular spikes recorded from cerebellar Purkinje cells. Conclusions: Our method could be expected to contribute to research in information processing in the cerebellar cortex and the development of a fully automatic spike-sorting algorithm by providing ground-truth data useful for deep learning. Highlights: A spike-sorting algorithm on hierarchical clustering was developed. It was applied to extracellular recordings from cerebellar Purkinje cells. Complex and simple spikes were discriminated based on their waveforms. Spike-sorting performance was quantified using in vivo Ca ²⁺ imaging data. The algorithm isolated over 96.6% of complex spikes.

Summary: Sensory processing is essential for motor control. Climbing fibers from the inferior olive transmit sensory signals to Purkinje cells, but how the signals are represented in the cerebellar cortex remains elusive. We examined the olivocerebellar organization of the mouse brain by optically measuring complex spikes (CSs) evoked by climbing fiber inputs over the entire dorsal surface of the cerebellum. We discovered that the surface was divided into approximately 200 segments each composed of ∼100 Purkinje cells that fired CSs synchronously. Our in vivo imaging of evoked responses revealed that whereas stimulation of four limb muscles individually similar global CS responses across nearly all segments, the timing and location of a stimulus were derived by Bayesian inference from coordinated activation and inactivation of multiple segments on a single trial basis. Our findings suggest that the cerebellum performs segment-based distributed population coding by assembling probabilistic sensory representation in individual segments.

Inositol 1,4,5-trisphosphate (InsP$_{3}$) is a second messenger that releases Ca$^{2+}$ from the intracellular stores. The InsP$_{3}$ receptor (InsP$_{3}$-R) was purified and its cDNA was cloned. We have found that InsP$_{3}$-R is identical to the P$_{400}$ protein identified as a protein enriched in the cerebellar Purkinje cells. We generated an L fibroblast cell transfectant that produced cDNA derived InsP$_{3}$-R. The expressed protein displays high affinity and specificity for InsP$_{3}$. InsP$_{3}$ induces Ca$^{2+}$ release from the membrane vesicles of the transfected cells. Incorporation of purified InsP$_{3}$-R into a lipid bilayer showed InsP$_{3}$ induced Ca$^{2+}$ release. These result suggest that InsP$_{3}$-R is a Ca$^{2+}$ release channel. Immunogold method using monoclonal antibodies against the receptor showed that it is highly condensed on the smooth surfaced endoplasmic reticulum (ER) and slightly on the outer nuclear membrane and rough ER. Cross linking experiments show that the InsP$_{3}$-R forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila melanogaster, and this region has the critical sequences for InsP$_{3}$ binding. We found novel subtypes of the InsP$_{3}$-R resulting from RNA-splicing that are expressed in a tissue-specific and developmentally specific manner and also resulting from different genes. It is believed that there are two Ca$^{2+}$ release mechanisms, InsP$_{3}$-induced Ca$^{2+}$ release (IICR) and Ca$^{2+}$-induced Ca$^{2+}$ release (CICR). Eggs are good materials to analyse the machanism of Ca$^{2+}$ signalling: fertilized hamster eggs exhibit repetitive Ca$^{2+}$ transients as well as the Ca$^{2+}$ wave. A monoclonal antibody to the InsP$_{3}$-R inhibited both IICR and CICR respectively upon injection of InsP$_{3}$ and Ca$^{2+}$ into hamster eggs. The antibody completely blocked sperm-induced Ca$^{2+}$ waves and Ca$^{2+}$ oscillations. The results indicate that Ca$^{2+}$ release in fertilized hamster eggs is mediated solely by the InsP$_{3}$-R, and that Ca$^{2+}$-sensitized IICR, but not CICR, generates Ca$^{2+}$ waves and Ca$^{2+}$ oscillations.