Hiroshi Kawasaki

The mammalian cerebrum has changed notably during evolution, with increases in neurons and glial cells accompanied by its expansion and folding. Although these evolutionary changes are thought to be crucial for the acquisition of higher cognitive functions, the molecular and cellular mechanisms underlying the development and evolution of the mammalian cerebrum are still not fully understood. This is partly because of the difficulty in analyzing these mechanisms using mice only. To overcome this problem, genetic manipulation techniques for the cerebrum of gyrencephalic carnivore ferrets have been established. Gene knockout in the ferret cerebrum has also been achieved using the CRISPR/Cas9 system. In this review, we summarize recent research into the mechanisms underlying the development and evolution of the cerebrum using ferrets.

Context: Diabetes-associated metabolic stress and anxiety reciprocally influence one another's onset and course. We previously linked excessive selenoprotein P (SeP, encoded by SELENOP in humans) to pathological conditions frequently observed in individuals with diabetes. Objective: The present study aimed to clarify the role of SeP in the metabolic stress-induced anxiety. Methods: We visualized Selenop expression in the mouse brain section via RNAscope in situ hybridization and used RT-qPCR to evaluate gene expression in brain regions. We created brain-specific Selenop knockout (bSelenop-/-) mice by mating Selenop-flox and Nestin-Cre mice and conducted behavior tests for anxiety-like behavior and spatial memory under both a standard (STD) and high-fat, high-sucrose diet (HFHSD) conditions. In a cross-sectional general population cohort study, we examined differences in serum selenoprotein P concentrations between individuals with and without anxiety symptoms. Results: RNAscope in situ hybridization identified glial and endothelial cells as the sources of SeP synthesis in the brain. Selenop was expressed at the same level in the brains of mice fed with an STD and HFHSD. bSelenop-/- mice did not exhibit altered body weight or glucose tolerance associated with HFHSD feeding. High-fat, high-sucrose diet aggravated the anxiety-like behavior in the control mice, whereas Selenop deletion in the brain ameliorated the anxiety-like behavior without affecting spatial memory. Epidemiological data revealed that serum selenoprotein P was significantly higher in subjects with anxiety symptoms. Conclusion/interpretation: These findings suggest that excess SeP production may be a common trait linking metabolic stress with anxiety.

Our understanding of the process of formation of gyri (ridges) and sulci (furrows) in the cerebrum during development is moving beyond the role of neurons. Glial cells such as astrocytes, which are the most common cell type in the brain, are especially prominent under the gyri and have been shown to be essential for gyrification in ferrets. Their dysfunction has been linked to a host of neurodevelopmental diseases and disorders in humans, leading to abnormal folding patterns, hence, it is crucial to understand their role in the mechanics of folding. In this work, we propose two hypotheses of how astrocytes affect cortical folding. Our previous study demonstrated that astrocytes proliferate in the white matter (subcortex), and that inhibiting this process impairs gyrification. This leads to the ‘ pushing ’ hypothesis, where astrocytes push up the cortex outwards, leading to formation of folds. On the other hand, ex vivo studies demonstrate areas of the cortex and subcortex that experience tension, due to the differential growth between materials in the brain tissue. This leads to the ‘ pulling ’ hypothesis, where astrocytes experience tension from the surrounding tissue, leading to their proliferation, distribution of tensile stresses, and initiation of growth in those regions. Using the theory of finite growth, we implement these hypotheses via morphogenetic growth ( pushing ) and stress-driven ( pulling ) growth criteria. We find that morphological trends during development between the pushing and pulling effects are not dissimilar, with a comparable gyrification index. The stress distributions from both models also show common features, but the pulling effect shows tension in the subcortex, which matches trends observed in experiments. Therefore, it is more likely that the astrocytes affect gyrification by proliferating as a response to tensile cues and decrease the tension they experience, leading to deeper folds, rather than astrocytes independently proliferating under a gyri and then pushing the cortex up.

Background: The mammalian cerebrum has changed substantially during evolution. Neurons and glial cells have increased, and the cerebrum has expanded and folded. Although these evolutionary changes are believed to be important for acquiring higher cognitive functions, the molecular mechanisms underlying the development and evolution of the mammalian cerebrum are not fully understood. This is partially due to the difficulty in analyzing these mechanisms using only mice. Summary: To overcome this limitation, we developed genetic manipulation techniques for the cerebrum of gyrencephalic carnivore ferrets. Gene knockout in the ferret cerebrum was achieved using the CRISPR/Cas9 system. Key Messages: This review highlights recent research from our lab and others on the mechanisms underlying the development and evolution of cortical folds using ferrets.

The functional significance of the morphological and genetic changes that occurred in the brain during evolution is not fully understood. Here we show the relationships between evolutionary changes of the brain and glymphatic circulation. We establish a mathematical model to simulate glymphatic circulation in the cerebral hemispheres, and our results show that cortical neurons accumulate in areas of the cerebral hemispheres where glymphatic circulation is highly efficient. We also find that cortical folds markedly enhance the efficiency of glymphatic circulation in the cerebral hemispheres. Furthermore, our in vivo study using ferrets reveals sulcus-dominant cerebrospinal fluid (CSF) influx, which enhances the efficiency of glymphatic circulation in the enlarged cerebral hemispheres of gyrencephalic brains. Sulcus-dominant CSF influx is mediated by preferential expression of aquaporin-4 in sulcal regions, and similar expression patterns of aquaporin-4 are also found in human cerebral hemispheres. These results indicate that evolutionary changes in the cerebral hemispheres are related to improved efficiency of glymphatic circulation. It seems plausible that the efficiency of glymphatic circulation is an important factor determining the evolutionary trajectory of the cerebral hemispheres.

Astrocytes in the cerebrum play important roles such as the regulation of synaptic functions, homeostasis, water transport, and the blood–brain barrier. It has been proposed that astrocytes in the cerebrum acquired diversity and developed functionally during evolution. Here, we show that like human astrocytes, ferret astrocytes in the cerebrum exhibit various morphological subtypes which mice do not have. We found that layer 1 of the ferret cerebrum contained not only protoplasmic astrocytes but also pial interlaminar astrocytes and subpial interlaminar astrocytes. Morphologically polarized astrocytes, which have a long unbranched process, were found in layer 6. Like human white matter, ferret white matter exhibited four subtypes of astrocytes. Furthermore, our quantification showed that ferret astrocytes had a larger territory size and a longer radius length than mouse astrocytes. Thus, our results indicate that, similar to the human cerebrum, the ferret cerebrum has a well‐developed diversity of astrocytes. Ferrets should be useful for investigating the molecular and cellular mechanisms leading to astrocyte diversity, the functions of each astrocyte subtype and the involvement of different astrocyte subtypes in various neurological diseases.

The mammalian cerebrum has changed substantially during evolution, characterized by increases in neurons and glial cells and by the expansion and folding of the cerebrum. While these evolutionary alterations are thought to be crucial for acquiring higher cognitive functions, the molecular mechanisms underlying the development and evolution of the mammalian cerebrum remain only partially understood. This is, in part, because of the difficulty in analyzing these mechanisms using mice only. To overcome this limitation, genetic manipulation techniques for the cerebrum of gyrencephalic carnivore ferrets have been developed. Furthermore, successful gene knockout in the ferret cerebrum has been accomplished through the application of the CRISPR/Cas9 system. This review mainly highlights recent research conducted using gyrencephalic carnivore ferrets to investigate the mechanisms underlying the development and evolution of cortical folds.