Agonist-independent GPCR activity regulates anterior-posterior targeting of olfactory sensory neurons.

G-protein-coupled receptors (GPCRs) are known to possess two different conformations, active and inactive, and they spontaneously alternate between the two in the absence of ligands. Here, we analyzed the agonist-independent GPCR activity for its possible role in receptor-instructed axonal projection. We generated transgenic mice expressing activity mutants of the ß2-adrenergic receptor, a well-characterized GPCR with the highest homology to odorant receptors (ORs). We found that mutants with altered agonist-independent activity changed the transcription levels of axon-targeting molecules--e.g., Neuropilin-1 and Plexin-A1--but not of glomerular segregation molecules--e.g., Kirrel2 and Kirrel3--thus causing shifts in glomerular locations along the anterior-posterior (A-P) axis. Knockout and in vitro experiments demonstrated that Gs, but not Golf, is responsible for mediating the agonist-independent GPCR activity. We conclude that the equilibrium of conformational transitions set by each OR is the major determinant of expression levels of A-P-targeting molecules.

Sequential arrival and graded secretion of Sema3F by olfactory neuron axons specify map topography at the bulb.

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) roughly correlate with their axonal projection sites along the dorsal-ventral (D-V) axis of the olfactory bulb (OB). Here we report that an axon guidance receptor, Neuropilin-2 (Nrp2), and its repulsive ligand, Semaphorin-3F (Sema3F), are expressed by OSNs in a complementary manner that is important for establishing olfactory map topography. Sema3F is secreted by early-arriving axons of OSNs and is deposited at the anterodorsal OB to repel Nrp2-positive axons that arrive later. Sequential arrival of OSN axons as well as the graded and complementary expression of Nrp2 and Sema3F by OSNs help to form the topographic order along the D-V axis.

Roles of odorant receptors during olfactory glomerular map formation.

The organization of the olfactory glomerular map involves the convergence of olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) into glomeruli in the olfactory bulb (OB). A remarkable feature of the olfactory glomerular map formation is that the identity of OR instructs the topography of the bulb, resulting in thousands of discrete glomeruli in mice. Several lines of evidence indicate that ORs control the expression levels of various kinds of transmembrane proteins to form glomeruli at appropriate regions of the OB. In this review, we will discuss how the OR identity is decoded by OSNs into gene expression through intracellular regulatory mechanisms.

Generation of Dopamine Transporter (DAT)-mCherry Knock-in Rats by CRISPR-Cas9 Genome Editing.

Midbrain dopaminergic neurons respond to rewards and have a crucial role in positive motivation and pleasure. Electrical stimulation of dopaminergic neurons and/or their axonal fibers and arborization has been often used to motivate animals to perform cognitive tasks. Still, the electrical stimulation is incompatible with electrophysiological recordings. In this light, optical stimulation following artificial expression of channelrhodopsin-2 (ChR2) in the cell membrane has been also used, but the expression level of ChR2 varies among researchers. Thus, we attempted to stably express ChR2 fused with a red fluorescence protein, mCherry, in dopaminergic neurons. Since dopamine transporter (DAT) gene is known as a marker for dopaminergic neurons, we inserted ChR2-mCherry into the downstream of the DAT gene locus of the rat genome by clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) genome editing and created DAT-ChR2-mCherry knock-in rats. Immunohistochemistry showed that ChR2-mCherry was expressed in dopaminergic neurons in homozygote knock-in rats, whereas whole-cell recordings revealed that ChR2-mCherry-positive neurons did not fire action potentials upon blue light stimulation, indicating that ChR2 was not functional for optogenetics. Nevertheless, fluorescent labeling of dopaminergic neurons mediated by mCherry could help characterize them physiologically and histologically.

Rice Putative Pectin Methyltransferase Gene OsPMT10 Is Required for Maintaining the Cell Wall Properties of Pistil Transmitting Tissues via Pectin Modification.

Precise directional control of pollen tube growth via mechanical guidance by pistil tissue is critical for the successful fertilization of flowering plants and requires active cell-to-cell communication and maintenance of softness in the transmitting tissue. However, the regulation of transmitting tissue softness as controlled by cell wall properties, especially pectin, has not been reported. Here we report that regulation of pectin methylesterification supports pollen elongation through pistil transmitting tissues in Oryza sativa. The rice pectin methylesterase gene OsPMT10 was strongly expressed in reproductive tissues, especially the pistil. The ospmt10 mutant did not have a significant effect on vegetative growth, but the fertility rate was reduced by approximately half. In the ospmt10 mutant, pollen tube elongation was observed in the transmitting tissue of the style, but approximately half of the pollen tubes did not extend all the way to the ovule. Tissue cross-sections of the upper ovary were prepared, and immunohistochemical staining using LM19 and LM20 showed that methylesterified pectin distribution was decreased in ospmt10 compared with the wild type. The decreased expression of methylesterified pectins in ospmt10 may have resulted in loss of fluidity in the apoplast space of the transmitting tissue, rendering it difficult for the pollen tube to elongate in the transmitting tissue and thereby preventing it from reaching the ovule.

Structural Alteration of Rice Pectin Affects Cell Wall Mechanical Strength and Pathogenicity of the Rice Blast Fungus Under Weak Light Conditions.

Pectin, a component of the plant cell wall, is involved in cell adhesion and environmental adaptations. We generated OsPG-FOX rice lines with little pectin due to overexpression of the gene encoding a pectin-degrading enzyme [polygalacturonase (PG)]. Overexpression of OsPG2 in rice under weak light conditions increased the activity of PG, which increased the degradation of pectin in the cell wall, thereby reducing adhesion. Under weak light conditions, the overexpression of OsPG decreased the pectin content and cell adhesion, resulting in abnormally large intercellular gaps and facilitating invasion by the rice blast fungus. OsPG2-FOX plants had weaker mechanical properties and greater sensitivity to biotic stresses than wild-type (WT) plants. However, the expression levels of disease resistance genes in non-infected leaves of OsPG2-FOX were more than twice as high as those of the WT and the intensity of disease symptoms was reduced, compared with the WT. Under normal light conditions, overexpression of OsPG2 decreased the pectin content, but did not affect cell adhesion and sensitivity to biotic stresses. Therefore, PG plays a role in regulating intercellular adhesion and the response to biotic stresses in rice.

Sharp wave-associated activity patterns of cortical neurons in the mouse piriform cortex.

The olfactory piriform cortex (PC) is thought to participate in olfactory associative memory. Like the hippocampus, which is essential for episodic memory, it belongs to an evolutionally conserved paleocortex and comprises a three-layered cortical structure. During slow-wave sleep, the olfactory PC becomes less responsive to external odor stimuli and instead displays sharp wave (SPW) activity similar to that observed in the hippocampus. Neural activity patterns during hippocampal SPW have been intensively studied in terms of memory consolidation; however, little is known about the activity patterns of olfactory cortical neurons during olfactory cortex sharp waves (OC-SPWs). In this study, we recorded multi-unit neural activities in the anterior PC in urethane-anesthetized mice. We found that the activity patterns of olfactory cortical neurons during OC-SPWs were non-randomly organized. Individual olfactory cortical neurons varied in the timings of their peak firing rates during OC-SPW events. Moreover, specific pairs of olfactory cortical neurons were more frequently activated together than expected by chance. On the basis of these observations, we speculate that coordinated activation of specific subsets of olfactory cortical neurons repeats during OC-SPWs, thereby facilitating synaptic plasticity underlying the consolidation of olfactory associative memories.

Differential expression of axon-sorting molecules in mouse olfactory sensory neurons.

In the mouse olfactory system, the axons of olfactory sensory neurons that express the same type of odorant receptor (OR) converge to a specific set of glomeruli in the olfactory bulb (OB). It is widely accepted that expressed OR molecules instruct glomerular segregation by regulating the expression of axon-sorting molecules. Although the relationship between the expression of axon-sorting molecules and OR types has been analyzed in detail, those between the expressions of axon-sorting molecules remain to be elucidated. Here we collected the expression profiles of four axon-sorting molecules from a large number of glomeruli in the OB. These molecules demonstrated position-independent mosaic expressions, but their patterns were not identical in the OB. Comparing their expressions identified positive and negative correlations between several pairs of genes even though they showed various expressions. Furthermore, the principal component analysis revealed that the factor loadings in the principal component 1, which explain the largest amount of variation, were most likely to reflect the degree of the cyclic nucleotide-gated (CNG) channel dependence on the expression of axon-sorting molecules. Thus, neural activity generated through the CNG channel is a major component in the generation of a wide variety of expressions of axon-sorting molecules in glomerular segregation.

Neural map formation in the mouse olfactory system.

In the mouse olfactory system, odorants are detected by ~1,000 different odorant receptors (ORs) produced by olfactory sensory neurons (OSNs). Each OSN expresses only one functional OR species, which is referred to as the "one neuron-one receptor" rule. Furthermore, OSN axons bearing the same OR converge to a specific projection site in the olfactory bulb (OB) forming a glomerular structure, i.e., the "one glomerulus-one receptor" rule. Based on these basic rules, binding signals of odorants detected by OSNs are converted to topographic information of activated glomeruli in the OB. During development, the glomerular map is formed by the combination of two genetically programmed processes: one is OR-independent projection along the dorsal-ventral axis, and the other is OR-dependent projection along the anterior-posterior axis. The map is further refined in an activity-dependent manner during the neonatal period. Here, we summarize recent progress of neural map formation in the mouse olfactory system.

Shaping the olfactory map: cell type-specific activity patterns guide circuit formation.

The brain constructs spatially organized sensory maps to represent sensory information. The formation of sensory maps has traditionally been thought to depend on synchronous neuronal activity. However, recent evidence from the olfactory system suggests that cell type-specific temporal patterns of spontaneous activity play an instructive role in shaping the olfactory glomerular map. These findings challenge traditional views and highlight the importance of investigating the spatiotemporal dynamics of neural activity to understand the development of complex neural circuits. This review discusses the implications of new findings in the olfactory system and outlines future research directions.

Top-down brain circuits for operant bradycardia.

Heart rate (HR) can be voluntarily regulated when individuals receive real-time feedback. In a rat model of HR biofeedback, the neocortex and medial forebrain bundle were stimulated as feedback and reward, respectively. The rats reduced their HR within 30 minutes, achieving a reduction of approximately 50% after 5 days of 3-hour feedback. The reduced HR persisted for at least 10 days after training while the rats exhibited anxiolytic behavior and an elevation in blood erythrocyte count. This bradycardia was prevented by inactivating anterior cingulate cortical (ACC) neurons projecting to the ventromedial thalamic nucleus (VMT). Theta-rhythm stimulation of the ACC-to-VMT pathway replicated the bradycardia. VMT neurons projected to the dorsomedial hypothalamus (DMH) and DMH neurons projected to the nucleus ambiguus, which innervates parasympathetic neurons in the heart.

HLA investigation in ICI-induced T1D and isolated ACTH deficiency including meta-analysis.

OBJECTIVE: Widespread use of immune checkpoint inhibitors (ICIs) in cancer treatment has led to an increase in the number of reported cases of immunotherapy-related endocrinopathies. This study aimed to analyze and compare human leukocyte antigen (HLA) signatures associated with ICI-induced type 1 diabetes (ICI-T1D) and isolated adrenocorticotropic hormone deficiency (ICI-IAD) in patients with both conditions. METHODS: HLA signatures were examined for their frequencies of occurrence in 22 patients with ICI-T1D without concurrent IAD, including 16 patients from nationwide reports (ICI-T1D group) and 14 patients with ICI-IAD without concurrent T1D (ICI-IAD group). The HLA signatures were also compared for their respective frequencies in 11 patients with ICI-T1D and ICI-IAD, including eight from nationwide reports (ICI-T1D/IAD group). RESULTS: In the ICI-T1D group, HLA-DRB1*09:01-DQB1*03:03 and DQA1*03:02, which are in linkage disequilibrium with DRB1*09:01-DQB1*03:03 and DRB1*13:02-DQB1*06:04, were susceptible to ICI-T1D, whereas DRB1*15:02-DQB1*06:01 was protective against ICI-T1D. In the ICI-IAD group, DPB1*09:01, C*12:02-B*52:01, and DRB1*15:02-DRB1*06:01, which are in strong linkage disequilibrium, were associated with susceptibility to ICI-IAD. Moreover, DRB1*15:02-DRB1*06:01 was not detected in the ICI-T1D/IAD group. CONCLUSIONS: This study revealed specific HLA signatures associated with ICI-T1D and ICI-IAD. Moreover, HLA-DRB1*15:02-DRB1*06:01, an ICI-IAD-susceptible HLA haplotype, coincides with the ICI-T1D-protective HLA haplotype, suggesting that the presence of DRB1*15:02-DRB1*06:01 may protect against the co-occurrence of T1D in patients with ICI-IAD.

Maintenance of Methyl-Esterified Pectin Level in Pollen Mother-Cell Stages Is Required for Microspore Development.

Pectin modification and degradation are vital for plant development, although the underlying mechanisms are still not well understood. Furthermore, reports on the function of pectin in early pollen development are limited. We generated OsPME-FOX rice lines with little methyl-esterified pectin even in the early-pollen mother-cell stage due to overexpression of the gene encoding pectin-methylesterase. Overexpression of OsPME1 in rice increased the activity of PME, which decreased the degree of pectin methyl esterification in the cell wall. OsPME1-FOX grew normally and showed abnormal phenotypes in anther and pollen development, especially in terms of the pollen mother-cell stage. In addition, we examined modifications of cell-wall polysaccharides at the cellular level using antibodies against polysaccharides. Immunohistochemical staining using LM19 and LM20 showed that methyl-esterified pectin distribution and the pectin contents in pollen mother-cell wall decreased in OsPME1-FOX compared with the wild type. Thus, the maintenance of methyl-esterified pectin plays a role in degrading and maintaining the pollen mother-cell wall during microspore development.

Modification and Characterization of Interfacial Bonding for Thermal Management of Ruthenium Interconnects in Next-Generation Very-Large-Scale Integration Circuits.

Ruthenium may replace copper interconnects in next-generation very-large-scale integration (VLSI) circuits. However, interfacial bonding between Ru interconnect wires and surrounding dielectrics must be optimized to reduce thermal boundary resistance (TBR) for thermal management. In this study, various adhesion layers are employed to modify bonding at the Ru/SiO2 interface. The TBRs of film stacks are measured using the frequency-domain thermoreflectance technique. TiN and TaN with high nitrogen contents significantly reduce the TBR of the Ru/SiO2 interface compared to common Ti and Ta adhesion layers. The adhesion layer thickness, on the other hand, has only minor effect on TBR when the thickness is within 2-10 nm. Hard X-ray photoelectron spectroscopy of deeply buried layers and interfaces quantitatively reveals that the decrease in TBR is attributed to the enhanced bonding of interfaces adjacent to the TaN adhesion layer, probably due to the electron transfer between the atoms at two sides of the interface. Simulations by a three-dimensional electrothermal finite element method demonstrate that decreasing the TBR leads to a significantly smaller temperature increase in the Ru interconnects. Our findings highlight the importance of TBR in the thermal management of VLSI circuits and pave the way for Ru interconnects to replace the current Cu-based ones.

Brain-wide mapping of presynaptic inputs to basolateral amygdala neurons.

The basolateral amygdala (BLA), a region critical for emotional processing, is the limbic hub that is connected with various brain regions. BLA neurons are classified into different subtypes that exhibit differential projection patterns and mediate distinct emotional behaviors; however, little is known about their presynaptic input patterns. In this study, we employed projection-specific monosynaptic rabies virus tracing to identify the direct monosynaptic inputs to BLA subtypes. We found that each neuronal subtype receives long-range projection input from specific brain regions. In contrast to their specific axonal projection patterns, all BLA neuronal subtypes exhibited relatively similar input patterns. This anatomical organization supports the idea that the BLA is a central integrator that associates sensory information in different modalities with valence and sends associative information to behaviorally relevant brain regions.

Cell type-specific patterned neural activity instructs neural map formation in the mouse olfactory system.

The development of precise neural circuits is initially directed by genetic programming and subsequently refined by neural activity. In the mouse olfactory system, axons from various olfactory sensory neurons expressing the same olfactory receptor converge onto a few spatially invariant glomeruli, generating the olfactory glomerular map in the olfactory bulbs. Using the glomerular map formation as a model, this review summarizes the current understanding of mechanisms underlying topographic map development in the mouse olfactory system and highlights how neural activity instructs the map refinement process.

Correction: Possible requirement of executive functions for high performance in soccer.

[This corrects the article DOI: 10.1371/journal.pone.0201871.].

Tb3+-doped fluorescent glass for biology.

Optical investigation and manipulation constitute the core of biological experiments. Here, we introduce a new borosilicate glass material that contains the rare-earth ion terbium(III) (Tb3+), which emits green fluorescence upon blue light excitation, similar to green fluorescent protein (GFP), and thus is widely compatible with conventional biological research environments. Micropipettes made of Tb3+-doped glass allowed us to target GFP-labeled cells for single-cell electroporation, single-cell transcriptome analysis (Patch-seq), and patch-clamp recording under real-time fluorescence microscopic control. The glass also exhibited potent third harmonic generation upon infrared laser excitation and was usable for online optical targeting of fluorescently labeled neurons in the in vivo neocortex. Thus, Tb3+-doped glass simplifies many procedures in biological experiments.

Rice Putative Methyltransferase Gene OsPMT16 Is Required for Pistil Development Involving Pectin Modification.

Pectin synthesis and modification are vital for plant development, although the underlying mechanisms are still not well understood. Furthermore, reports on the function of pectin in the pistil are limited. Herein, we report the functional characterization of the OsPMT16 gene, which encodes a putative pectin methyltransferase (PMT) in rice. The cell walls of rice leaves contain less pectin, and chemical analysis of pectin in the flower organ had not been previously performed. Therefore, in the present study, the amount of pectin in the reproductive tissues of rice was investigated. Of the reproductive tissues, the pistil was especially rich in pectin; thus, we focused on the pistil. OsPMT16 expression was confirmed in the pistil, and effects of pectin methylesterification regulation on the reproductive stage were investigated by studying the phenotype of the T-DNA insertion mutant. The ospmt16 mutant showed significantly reduced fertility. When the flowers were observed, tissue morphogenesis was abnormal in the pistil. Immunofluorescence staining by pectin-specific monoclonal antibodies of the pistil revealed that total pectin and esterified pectin were decreased among ospmt16 mutants. These results indicate that OsPMT16 contributes significantly to pistil development during reproductive growth.

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution.

Structural elucidation and molecular scrutiny of cerebral vasculature is crucial for understanding the functions and diseases of the brain. Here, we introduce SeeNet, a method for near-complete three-dimensional visualization of cerebral vascular networks with high signal-to-noise ratios compatible with molecular phenotyping. SeeNet employs perfusion of a multifunctional crosslinker, vascular casting by temperature-controlled polymerization of hybrid hydrogels, and a bile salt-based tissue-clearing technique optimized for observation of vascular connectivity. SeeNet is capable of whole-brain visualization of molecularly characterized cerebral vasculatures at the single-microvessel level. Moreover, SeeNet reveals a hitherto unidentified vascular pathway bridging cerebral and hippocampal vessels, thus serving as a potential tool to evaluate the connectivity of cerebral vasculature.