Dissecting amygdala cell types in fear and extinction

The mammalian brain consists of billions of neurons; Individual neurons serve as building blocks (Cajal 1911, translation Swanson and Swanson 1995). However, studying individual neurons is simply insufficient to understand how the brain works. A promising approach is the cell-type-specific approach, an effort to classify neurons that perform the same function as a single cell type (functional definition, see (Luo et al., 2008)) and to understand their roles in information processing and behavioral outputs. Nevertheless, the limitation of this definition is that we barely know the precise functions or roles of neurons, and even in very well-characterized neurons such as retinal ganglion cells, there would likely be remaining unknown functions. Thus, as an operational definition to drive neuroscience forward, defining cell types using genetic tools that allow us to access specific subsets of neurons was suggested and widely accepted in an almost implicit manner. This consensus is based on an optimistic view that, at some point, the operational genetic definition and the ultimate functional definition would converge. In this thesis, having this philosophy in mind, I try to match several operationally defined amygdala cell types with their distinct functions/roles in the context of fear and extinction learning. In Project 1, I demonstrate that a cell-type in the amygdala complex defined by molecular marker expression exerts essential functions in fear and extinction by composing a unique mutual inhibition circuit motif. In Project 2, I find that a cell-type in the basolateral amygdala defined by di-synaptic downstream target show unprecedented functional specificity in fear learning. Finally, in Project 3, I aim to characterize functions and roles of cell types in the basolateral amygdala defined by dynamic, neuronal activity-dependent gene expression upon learning. Collectively, this thesis serves as an important stepping stone to achieving the convergence between definitions of a cell type

Stability of crystallographic texture in laser powder bed fusion: Understanding the competition of crystal growth using a single crystalline seed

In metal additive manufacturing, crystallographic orientation control is a promising method for tailoring the functions of metallic parts. However, despite its importance in the fabrication of texture-controlled functional parts, the stability of the crystallographic texture is not widely discussed. Herein, the crystallographic texture stability under laser powder bed fusion was investigated. Two methodologies were employed. One is that a laser scanning strategy was alternately changed for a specific number of layers. The other is a “seeding” experiment in which single-crystalline substrates with controlled crystallographic orientations in the building (z-) direction and the xy-plane (perpendicular to the building direction) were used as the starting substrate. The transient zone width, where the crystallographic orientation was inherited from the layer beneath, was analyzed to evaluate the texture stability. The crystallographic direction of the seed within the xy-plane, rather than the building direction, determined the transient zone width, i.e., the texture stability. In particular, the texture in the newly deposited portion was stable when the laser scanning direction matched the orientation in the underneath layer, otherwise the crystal orientation switched rapidly, such that the orientation was parallel to the scanning direction. Interestingly, the crystallographic orientation along the building direction in the underneath layer hardly impacted the stability of the texture. Therefore, for the first time, it has been clarified that the orientation in the scanning direction, rather than the building direction, was preferentially stabilized, whereas the orientation in the other directions secondary stabilized.Ishimoto T., Hagihara K., Hisamoto K., et al. Stability of crystallographic texture in laser powder bed fusion: Understanding the competition of crystal growth using a single crystalline seed. Additive Manufacturing, 43, 102004. https://doi.org/10.1016/j.addma.2021.102004

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Long-term down-regulation of GABA decreases orientation selectivity without affecting direction selectivity in mouse primary visual cortex

Inhibitory interneurons play important roles in the development of brain functions. In the visual cortex, functional maturation of inhibitory interneurons is essential for ocular dominance plasticity. However, roles of inhibitory interneurons in the development of orientation and direction selectivity, fundamental properties of primary visual cortex, are less understood. We examined orientation and direction selectivity of neurons in GAD67-GFP (Δneo) mice, in which expression of GABA in the brain is decreased in the newborn. We used in vivo two-photon calcium imaging to examine visual response of neurons in these mice and found that long-term decrease of GABA led to increase of response amplitude to non-preferred orientation of visual stimuli, which decreased orientation selectivity. In contrast, direction selectivity was not affected. These results suggest that orientation selectivity is decreased in mice with GABA down-regulation during development

Cell-Type-Specific Thalamocortical Inputs Constrain Direction Map Formation in Visual Cortex

Summary: Finding the relationship between individual cognitive functions and cell-type-specific neuronal circuits is a central topic in neuroscience. In cats, the lateral geniculate nucleus (LGN) contains several cell types carrying spatially and temporally precise visual information. Whereas LGN cell types lack selectivity for motion direction, neurons in the primary visual cortex (area 17) exhibit sharp direction selectivity. Whether and how such de novo formation of direction selectivity depends on LGN cell types remains unknown. Here, we addressed this question using in vivo two-photon calcium imaging in cat area 17, which consists of two compartments receiving different combinations of inputs from the LGN cell types. The direction map in area 17 showed unique fragmented organization and was present only in small and distributed cortical domains. Moreover, direction-selective domains preferentially localized in specific compartments receiving Y and W inputs carrying low spatial frequency visual information, indicating that cell-type-specific thalamocortical projections constrain the formation of direction selectivity. : Nishiyama et al. find fragmented organization of direction columns in the cat primary visual cortex. The direction columns are predominantly located in cortical domains preferring low spatial frequency. The results suggest that cell-type-specific thalamocortical projections from LGN may constrain direction column formation in the cat visual cortex. Keywords: brain mapping, in vivo two-photon imaging, functional columns, direction selectivity, thalamus, visual cortex, thalamocortical circui

Poly-l-gamma-glutamic acid production by recombinant Bacillus subtilis without pgsA gene

Abstract Poly-gamma-glutamic acid (PGA) is a promising bio-based polymer that shares many functions with poly (acrylic acid) and its derivatives. Thus, technologies for efficient production and molecular size control of PGA are required to expand the application of this useful biopolymer. In Bacillus strains, PGA is synthesized by the PgsBCA protein complex, which is encoded by the pgsBCA gene operon, otherwise is known as ywsC and ywtAB operons and/or capBCA operon. Hence, we investigated responsible components of the PgsBCA complex in B. subtilis for over-production of PGA. In particular, we constructed genomic pgsBCA gene-deletion mutants of B. subtilis. And also, we assembled high copy-number plasmids harboring σA-dependent promoter, leading to high-level expression of all combinations of pgsBCA, pgsBC, pgsBA, pgsCA, pgsB, pgsC, and/or pgsA genes. Subsequently, PGA production of the transformed B. subtilis mutant was determined in batch fermentation using medium supplemented with l-glutamate. PGA production by the transformants introduced with pgsBC genes (lacking the genomic pgsBCA genes) was 26.0 ± 3.0 g L−1, and the enantiomeric ratio of d- and l-glutamic acid (d/l-ratio) in the produced PGA was 5/95. In contrast, d/l-ratio of produced PGA by the transformants introduced with pgsBCA genes (control strains) was 75/25. In conclusion, B. subtilis without pgsA gene could over-produce PGA with an l-rich enantiomeric ratio