Organotypic Tissue Culture of Adult Rodent Retina Followed by Particle-Mediated Acute Gene Transfer In Vitro

BACKGROUND: Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age) which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames' medium (>26 mL) per retina, a higher speed (constant 55 rpm) of agitation by rotary shaker, and a greater concentration (10%) of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age). The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP) into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons. CONCLUSIONS/SIGNIFICANCE: This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing molecular biological bioassays that used to be conducted in isolated cultured cells

PLD4 Is Involved in Phagocytosis of Microglia: Expression and Localization Changes of PLD4 Are Correlated with Activation State of Microglia

Phospholipase D4 (PLD4) is a recently identified protein that is mainly expressed in the ionized calcium binding adapter molecule 1 (Iba1)-positive microglia in the early postnatal mouse cerebellar white matter. Unlike PLD1 and PLD2, PLD4 exhibits no enzymatic activity for conversion of phosphatidylcholine into choline and phosphatidic acid, and its function is completely unknown. In the present study, we examined the distribution of PLD4 in mouse cerebellar white matter during development and under pathological conditions. Immunohistochemical analysis revealed that PLD4 expression was associated with microglial activation under such two different circumstances. A primary cultured microglia and microglial cell line (MG6) showed that PLD4 was mainly present in the nucleus, except the nucleolus, and expression of PLD4 was upregulated by lipopolysaccharide (LPS) stimulation. In the analysis of phagocytosis of LPS-stimulated microglia, PLD4 was co-localized with phagosomes that contained BioParticles. Inhibition of PLD4 expression using PLD4 specific small interfering RNA (siRNA) in MG6 cells significantly reduced the ratio of phagocytotic cell numbers. These results suggest that the increased PLD4 in the activation process is involved in phagocytosis of activated microglia in the developmental stages and pathological conditions of white matter

Early Maternal and Social Deprivation Expands Neural Stem Cell Population Size and Reduces Hippocampus/Amygdala-Dependent Fear Memory.

Early life stress can exert detrimental or beneficial effects on neural development and postnatal behavior depending on the timing, duration, strength, and ability to control the stressors. In this study, we utilized a maternal and social deprivation (MSD) model to investigate the effects of early life stress on neural stem cells (NSCs) and neurogenesis in the adult brain. We found that MSD during the stress-hyporesponsive period (SHRP) (early-MSD), when corticosterone secretion is suppressed, increased the size of the NSC population, whereas the same stress beyond the SHRP abrogated these effects. Early-MSD enhanced neurogenesis not only in the dentate gyrus of the hippocampus, one of the classic neurogenic regions, but also in the amygdala. In addition, mice exposed to early-MSD exhibited a reduction in amygdala/hippocampus-dependent fear memory. These results suggest that animals exposed to early life stress during the SHRP have reinforced stress resilience to cope with perceived stressors to maintain a normal homeostatic state

A Myelin Galactolipid, Sulfatide, Is Essential for Maintenance of Ion Channels on Myelinated Axon But Not Essential for Initial Cluster Formation

Myelinated axons are divided into four distinct regions: the node of Ranvier, paranode, juxtaparanode, and internode, each of which is characterized by a specific set of axonal proteins. Voltage-gated N

Neuron-Glia Interaction in Neuroinflammation

X, 187 p. 25 illus., 24 illus. in color.online re

Microglia-induced activation of noncanonical Wnt signaling aggravates neurodegeneration in demyelinating disorders

Oligodendrocytes are myelinating cells of the central nervous system. Multiple sclerosis (MS) is a demyelinating disease characterized by both myelin loss and neuronal degeneration. However, the molecular mechanisms underlying neuronal degeneration in demyelinating disorders are not fully understood. In the experimental autoimmune encephalomyelitis (EAE) demyelinatingmouse model of MS, inflammatory microglia produce cytokines, including interleukin-1β (IL-1β). Since microglia and noncanonical Wnt signaling components in neurons, such as the coreceptor Ror2, were observed in the spinal cords of mice with EAE (EAE mice), we postulated that the interplay between activated microglia and spinal neurons under EAE conditions is mediated through noncanonical Wnt signaling. EAE treatment upregulated in vivo expression of noncanonical Wnt signaling components in spinal neurons through microglial activation. In accordance with the neuronal degeneration detected in the EAE spinal cord in vivo, coculture of spinal neurons with microglia or the application of recombinant IL-1β upregulated noncanonical Wnt signaling and induced neuron death, which was suppressed by the inhibition of the Wnt-Ror2 pathway. Ectopic noncanonical Wnt signaling aggravated the demyelinating pathology in another MS mouse model due to Wnt5a-induced neurodegeneration. The linkage between activated microglia and neuronal Wnt-Ror2 signaling may provide a candidate target for therapeutic approaches to demyelinating disorders