Differential contribution of canonical and noncanonical NLGN3 pathways to early social development and memory performance.

Neuroligin (NLGN) 3 is a postsynaptic cell adhesion protein organizing synapse formation through two different types of transsynaptic interactions, canonical interaction with neurexins (NRXNs) and a recently identified noncanonical interaction with protein tyrosine phosphatase (PTP) δ. Although, NLGN3 gene is known as a risk gene for neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID), the pathogenic contribution of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ pathways to these disorders remains elusive. In this study, we utilized Nlgn3 mutant mice selectively lacking the interaction with either NRXNs or PTPδ and investigated their social and memory performance. Neither Nlgn3 mutants showed any social cognitive deficiency in the social novelty recognition test. However, the Nlgn3 mutant mice lacking the PTPδ pathway exhibited significant decline in the social conditioned place preference (sCPP) at the juvenile stage, suggesting the involvement of the NLGN3-PTPδ pathway in the regulation of social motivation and reward. In terms of learning and memory, disrupting the canonical NRXN pathway attenuated contextual fear conditioning while disrupting the noncanonical NLGN3-PTPδ pathway enhanced it. Furthermore, disruption of the NLGN3-PTPδ pathway negatively affected the remote spatial reference memory in the Barnes maze test. These findings highlight the differential contributions of the canonical NLGN3-NRXN and noncanonical NLGN3-PTPδ synaptogenic pathways to the regulation of higher order brain functions associated with ASD and ID.

ILF3 prion-like domain regulates gene expression and fear memory under chronic stress.

The prion-like domain (PrLD) is a class of intrinsically disordered regions. Although its propensity to form condensates has been studied in the context of neurodegenerative diseases, the physiological role of PrLD remains unclear. Here, we investigated the role of PrLD in the RNA-binding protein NFAR2, generated by a splicing variant of the Ilf3 gene. Removal of the PrLD in mice did not impair the function of NFAR2 required for survival, but did affect the responses to chronic water immersion and restraint stress (WIRS). The PrLD was required for WIRS-sensitive nuclear localization of NFAR2 and WIRS-induced changes in mRNA expression and translation in the amygdala, a fear-related brain region. Consistently, the PrLD conferred resistance to WIRS in fear-associated memory formation. Our study provides insights into the PrLD-dependent role of NFAR2 for chronic stress adaptation in the brain.

Functional Evaluation of 3D Liver Models Labeled with Polysaccharide Functionalized Magnetic Nanoparticles.

Establishing a rapid in vitro evaluation system for drug screening is essential for the development of new drugs. To reproduce tissues/organs with functions closer to living organisms, in vitro three-dimensional (3D) culture evaluation using microfabrication technology has been reported in recent years. Culture on patterned substrates with controlled hydrophilic and hydrophobic regions (Cell-ableTM) can create 3D liver models (miniature livers) with liver-specific Disse luminal structures and functions. MRI contrast agents are widely used as safe and minimally invasive diagnostic methods. We focused on anionic polysaccharide magnetic iron oxide nanoparticles (Resovist®) and synthesized the four types of nanoparticle derivatives with different properties. Cationic nanoparticles (TMADM) can be used to label target cells in a short time and have been successfully visualized in vivo. In this study, we examined the morphology of various nanoparticles. The morphology of various nanoparticles showed relatively smooth-edged spherical shapes. As 3D liver models, we prepared primary hepatocyte-endothelial cell heterospheroids. The toxicity, CYP3A, and albumin secretory capacity were evaluated in the heterospheroids labeled with various nanoparticles. As the culture period progressed, the heterospheroids labeled with anionic and cationic nanoparticles showed lower liver function than non-labeled heterospheroids. In the future, there is a need to improve the method of creation of artificial 3D liver or to design a low-invasive MRI contrast agent to label the artificial 3D liver.

Effects of chronic fentanyl administration on behavioral characteristics of mice.

BACKGROUND: Fentanyl, a synthetic opioid categorized as a narcotic analgesic, has a 100- to 200-fold stronger effect than most opioids, such as morphine. Fatal accidents due to chronic use and abuse of fentanyl are a worldwide social problem. One reason for the abuse of fentanyl is its psychostimulant effects that could induce behavioral changes. The effects of chronic fentanyl administration on behavior, however, are unclear. METHODS: Adult male C57BL/6J mice were chronically administered fentanyl (0.03 or 0.3 mg/kg/d i.p.), and various behaviors were assessed using a behavioral test battery. RESULTS: Mice chronically administered a high dose of fentanyl (0.3 mg/kg/d) exhibited decreased anxiety-like behavior as assessed by the open field and elevated plus maze tests. On the other hand, interruption of fentanyl administration led to increased anxiety-like behavior as observed in the light and dark transition test. The hot plate test revealed that chronic administration of fentanyl reduced pain sensitivity. High-dose chronic fentanyl administration reduced the locomotor stimulatory effects of cocaine. The results, however, failed to reach the threshold for study-wide statistical significance. CONCLUSION: Chronic fentanyl administration induces some behavioral changes in mice. Although further studies are needed to clarify the underlying mechanisms of the behavioral effects of chronic fentanyl administration, our findings suggest that fentanyl is safe under properly controlled conditions.

Observation of Positively Charged Magnetic Nanoparticles Inside HepG2 Spheroids Using Electron Microscopy.

Magnetic resonance imaging (MRI) using magnetic nanoparticles has been used to diagnose vascular diseases as well as to monitor transplanted cells and tissues. In this study, we synthesized magnetic iron oxide nanoparticles (TMADM-03), electrically charged by the presence of a cationic end-group substitution of dextran, and observed these nanoparticles inside three-dimensional models of HepG2 spheroids, which mimic tissues. Patterned cell array glass disks were prepared to visualize the presence of TMADM-03 uptaken by HepG2 spheroids using transmission electron microscopy (TEM). The HepG2 cells (2 × 10(5) cells) were inoculated onto Cell-able™ 12-well plates. After 48 h of culture, the cells were incubated with 75 µg Fe/ml TMADM-03 in culture medium for 24 h. To investigate the cellular function of the HepG2 spheroids, the albumin secretion was evaluated by an ELISA. The albumin secretion after incubation for 24 h was reduced compared with the secretion prior to the addition of TMADM-03. TEM image samples were prepared in a planar direction or a vertical direction to the HepG2 spheroids on patterned cell array glass disks. The incorporation of TMADM-03 inside the HepG2 spheroids was confirmed. In addition, TMADM-03 could be observed in the deeper layers of the spheroids, and this was localized in the lysosomes. These data suggest that the novel magnetic iron oxide nanoparticles invade three-dimensional HepG2 spheroids.

Phenotypical and functional alterations during the expansion phase of invariant Valpha14 natural killer T (Valpha14i NKT) cells in mice primed with alpha-galactosylceramide.

Invariant Valpha14 natural killer T (Valpha14i NKT) cells are a unique immunoregulatory T-cell population that is restricted by CD1d. The glycolipid alpha-galactosylceramide (alpha-GalCer) is presented by CD1d and causes robust Valpha14i NKT-cell activation. Three days after injection of alpha-GalCer, Valpha14i NKT cells vigorously increase in number and then gradually decrease to normal levels. In the present study, we found that the re-administration of alpha-GalCer into mice primed 3 days earlier causes a marked increase in serum interleukin-4 and interferon-gamma. Intracellular staining revealed that the only expanded Valpha14i NKT cells are responsible for the enhanced cytokine production. The enhanced cytokine production was correlated with an increased number of Valpha14i NKT cells after priming. Additionally, primed Valpha14i NKT cells produced larger amounts of cytokine as compared with naive Valpha14i NKT cells when cultured with alpha-GalCer-pulsed dendritic cells. Thus, we considered that a subset of expanded Valpha14i NKT cells acquired a strong ability to produce cytokines. In contrast to mice primed 3 days earlier, cytokine production is markedly diminished in mice primed 7 days earlier. The expanded Valpha14i NKT cells altered the surface phenotype (NK1.1- CD69-) and contained intracellular interferon-gamma. Additionally, we found that primed Valpha14i NKT cells did not disappear or down-regulate surface TCR expression when re-injected with alpha-GalCer as compared with naive Valpha14i NKT cells. These results demonstrate that the function and surface phenotype of Valpha14i NKT cells is dramatically altered after alpha-GalCer priming.