Understanding the structural organization of organs and organisms at the cellular level is a fundamental challenge in biology. This task has been approached by reconstructing three-dimensional structure from images taken from serially sectioned tissues, which is not only labor-intensive and time-consuming but also error-prone. Recent advances in tissue clearing techniques allow visualization of cellular structures and neural networks inside of unsectioned whole tissues or the entire body. However, currently available protocols require long process times. Here, we present the rapid and highly reproducible ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) method that clears tissues or the whole body within 1 day while preserving tissue architecture and protein-based signals derived from endogenous fluorescent proteins. Moreover, ACT-PRESTO is compatible with conventional immunolabeling methods and expedites antibody penetration into thick specimens by applying pressure. The speed and consistency of this method will allow high-content mapping and analysis of normal and pathological features in intact organs and bodies.

Hereditary ataxic mice, tottering (tg) and rolling Nagoya (tg(rol)), carry mutations in the P/Q-type Ca(2+) channel alpha(1A) subunit gene. The positions of the mutations and the neurological phenotypes are known, but the mechanisms of how the mutations cause the symptoms and how the different mutations lead to various onset and severity have remained unsolved. Here we compared fundamental properties of excitatory synaptic transmission in the cerebellum and roles of Ca(2+) channel subtypes therein among wild-type control, tg, and tg(rol) mice. The amplitude of EPSC of the parallel fiber-Purkinje cell (PF-PC) synapses was considerably reduced in ataxic tg(rol). Although the amplitude of the parallel fiber-mediated EPSC was only mildly decreased in young non-ataxic tg mice, it was drastically diminished in adult ataxic tg mice of postnatal day 28-35, showing a good correlation between the impairment of the PF-PC synaptic transmission and manifestation of ataxia. In contrast, the EPSC amplitude of the climbing fiber-Purkinje cell (CF-PC) synapses was preserved in tg, and it was even increased in tg(rol), which was associated with altered properties of the postsynaptic glutamate receptors. The climbing fiber-mediated EPSC was more dependent on other Ca(2+) channel subtypes in mutant mice, suggesting that such compensatory mechanisms contribute to maintaining the CF-PC synaptic transmission virtually intact. The results indicate that different mutations of the P/Q-type Ca(2+) channel not only cause the primary effect of different severity but also lead to diverse additional secondary effects, resulting in disruption of well balanced neural networks.

Mitochondrial division is critical for the maintenance and regulation of mitochondrial function, quality and distribution. This process is controlled by cytosolic actin-based constriction machinery and dynamin-related protein 1 (Drp1) on mitochondrial outer membrane (OMM). Although mitochondrial physiology, including oxidative phosphorylation, is also important for efficient mitochondrial division, morphological alterations of the mitochondrial inner-membrane (IMM) have not been clearly elucidated. Here we report spontaneous and repetitive constriction of mitochondrial inner compartment (CoMIC) associated with subsequent division in neurons. Although CoMIC is potentiated by inhibition of Drp1 and occurs at the potential division spots contacting the endoplasmic reticulum, it appears on IMM independently of OMM. Intra-mitochondrial influx of Ca2+ induces and potentiates CoMIC, and leads to K+-mediated mitochondrial bulging and depolarization. Synergistically, optic atrophy 1 (Opa1) also regulates CoMIC via controlling Mic60-mediated OMM–IMM tethering. Therefore, we propose that CoMIC is a priming event for efficient mitochondrial division.

This study examined whether regular exercise training, at a level that would be recommended for middle-aged people interested in improving fitness could lead to improved cognitive performance and increased blood flow to the brain in another primate species. Adult female cynomolgus monkeys were trained to run on treadmills for one hour a day, 5 days a week, for a 5 month period (n=16; 1.9±0.4 miles/day). A sedentary control group sat daily on immobile treadmills (n=8). Half of the runners had an additional sedentary period for 3 months at the end of the exercise period (n=8). In all groups, half of the monkeys were middle-aged (10-12 years old) and half were more mature (15-17 years old). Starting the fifth week of exercise training, monkeys underwent cognitive testing using the Wisconsin General Testing Apparatus (WGTA). Regardless of age, the exercising group learned to use the WGTA significantly faster (4.6±3.4 days) compared to controls (8.3±4.8 days; p=0.05). At the end of 5 months of running monkeys showed increased fitness, and the vascular volume fraction in the motor cortex in mature adult running monkeys was increased significantly compared to controls (p=0.029). However, increased vascular volume did not remain apparent after a three-month sedentary period. These findings indicate that the level of exercise associated with improved fitness in middle-aged humans is sufficient to increase both the rate of learning and blood flow to the cerebral cortex, at least during the period of regular exercise.

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The cerebellum is involved in the learning and retention of motor skills. Using animal and human models, a number of studies have shown that long-term motor skill training induces structural and functional plasticity in the cerebellum. The aim of this study was to investigate whether macroscopic alteration in the volume of cerebellum occurs in basketball players who had learned complex motor skills and practiced them intensively for a long time. Three-dimensional magnetic resonance imaging volumetry was performed in basketball players (n = 19) and healthy controls (n = 20), and the volumes of cerebellum and vermian lobules were compared between two groups. Although there was no macroscopic plasticity detected in the cerebellum as a whole, detailed parcellation of cerebellum revealed morphological enlargement in the vermian lobules VI-VII (declive, folium, and tuber) of basketball players (P < 0.0166), which might then be interpreted as evidence for plasticity. This finding suggests that the extensive practice and performance of sports-related motor skills activate structural plasticity of vermian lobules in human cerebellum and suggests that vermian VI-VII plays an important role in motor learning.

A considerable number of new neurons are generated daily in the dentate gyrus (DG) of the adult hippocampus, but only a subset of these survive, as many adult-generated neurons undergo programmed cell death (PCD). However, the significance of PCD in the adult brain for the functionality of DG circuits is not known. Here we examined the electrophysiological and behavioral characteristics of Bax-KO mice in which PCD of post-mitotic neurons is prevented. The continuous increase in DG cell numbers in Bax-KO mice, resulted in the readjustment of afferent and efferent synaptic connections, represented by age-dependent reductions in the dendritic arborization of DG neurons and in the synaptic contact ratio of mossy fibers (MF) with CA3 dendritic spines. These neuroanatomical changes were associated with reductions in synaptic transmission and reduced performance in a contextual fear memory task in 6-month old Bax-KO mice. These results suggest that the elimination of excess DG neurons via Bax-dependent PCD in the adult brain is required for the normal organization and function of the hippocampus.

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