A new minimally-invasive method for microinjection into the mouse spinal dorsal horn.

Noninvasive gene delivery to the spinal dorsal horn (SDH) remains challenging because existing methods to directly microinject vectors require laminectomy, which leads to tissue damage and inflammation. Such responses might hamper accurate readouts of cellular and behavioural effects of an introduced gene. Here we develop a new minimally-invasive SDH microinjection technique without the need of laminectomy in which a microcapillary is inserted into the SDH parenchyma through an intervertebral space. Using this method, we microinjected adeno-associated virus with an astrocytic promoter into the SDH and achieved efficient gene expression in an astrocyte-specific manner without gliosis, neuronal loss or inflammation. Furthermore, astrocytic loss- and gain-of-function of the transcription factor STAT3 by expressing a dominant-negative form and a constitutive-active form of STAT3, respectively, demonstrated the necessity and sufficiency of astrocytic STAT3 in the maintenance of neuropathic pain following peripheral nerve injury, a debilitating chronic pain state in which currently available treatments are frequently ineffective. Thus, our technique enables manipulation of gene expression in cell type- and spatial-specific manners without adverse effects, and may be useful for research in SDH physiology and pathology.

Three dynamin-related protein 5B genes are related to plastid division in Physcomitrella patens.

Dynamin family proteins in eukaryotic cells assemble into rings or spirals on the surface of membranes and pinch the membranes. We found 21 dynamin-related protein (DRP) genes in the Physcomitrella patens genome. Phylogenetic analysis indicated that three of them (PpDRP5B-1, PpDRP5B-2, and PpDRP5B-3) showed robust monophyly with Arabidopsis thaliana DRP5B and Cyanidioschyzon merolae CmDnm2, both of which are related to plastid division. Quantitative RT-PCR analysis showed that the amounts of DRP5B-3 transcripts were 14-fold and 8-fold higher than those of DRP5B-1 and DRP5B-2, respectively. We generated PpDRP5B knockout transformants for each of these genes. Subapical protonemata cells in wild-type plants had an average of 47 chloroplasts. The cells in the PpDRP5B-3 knockout transformant had slightly enlarged chloroplasts, with an average chloroplast number of 28, whereas the PpDRP5B-1 and 5B-2 knockout lines had no effect on chloroplast number in P. patens. To analyze function of each PpDRP5B gene, we generated double- and triple-knockout lines. Whereas there were 32 chloroplasts in a cell of the PpDRP5B-1/5B-2 double-knockout lines, the triple-knockout line had only a few macrochloroplasts. A transient expression assay with the triple-knockout line demonstrated that the PpDRP5B-3 gene could recover the normal chloroplast phenotype.

Sustained activation of ERK signaling in astrocytes is critical for neuronal injury-induced monocyte chemoattractant protein-1 production in rat corticostriatal slice cultures.

We previously demonstrated that N-methyl-D-aspartate (NMDA) treatment (50 microM, 3 h) induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2), a CC chemokine implicated in ischemic and excitotoxic brain injury, in rat corticostriatal slice cultures. In this study, we investigated the signaling mechanisms for NMDA-induced MCP-1 production in slice cultures. The results showed a close correlation between NMDA-induced neuronal injury and MCP-1 production, and an abrogation of NMDA-induced MCP-1 production in NMDA-pretreated slices where neuronal cells had been eliminated. These results collectively indicate that NMDA-induced neuronal injury led to astrocytic MCP-1 production. NMDA-induced MCP-1 production was significantly inhibited by U0126, an inhibitor of extracellular signal-regulated kinase (ERK). Immunostaining for phosphorylated ERK revealed that transient neuronal ERK activation was initially induced and subsided within 30 min, followed by sustained ERK activation in astrocytes. Treatment with U0126 during only the early phase (U0126 was washed out at 15 or 30 min after NMDA administration) suppressed early activation of ERK in neuronal cells, but not later activation of ERK in astrocytes. In this case, MCP-1 production was not suppressed, suggesting that activation of neuronal ERK is not necessary for MCP-1 production. In contrast, delayed application of U0126 at 3 h after the beginning of NMDA treatment inhibited MCP-1 production to the same degree as that observed when U0126 was applied from 3 h before NMDA administration. These findings suggest that sustained activation of the ERK signaling pathway in astrocytes plays a key role in neuronal injury-induced MCP-1 production.