ABSTRACT
The neuron-specific K-Cl cotransporter (KCC2) is hypothesized to function as an active Cl- extrusion pathway important in postsynaptic inhibition mediated by ligand-gated anion channels, like gamma-aminobutyric acid type A (GABAA) and glycine receptors. To understand better the functional role of KCC2 in the nervous system, we developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to characterize and localize KCC2 in the rat cerebellum. The antibodies specifically recognized the KCC2 protein which is an approximately 140-kDa glycoprotein detectable only within the central nervous system. The KCC2 protein displayed a robust and punctate distribution in primary cultured retinal amacrine cells known to form exclusively GABAAergic synapses in culture. In immunolocalization studies, KCC2 was absent from axons and glia but was highly expressed at neuronal somata and dendrites, indicating a specific postsynaptic distribution of the protein. In the granule cell layer, KCC2 exhibited a distinct colocalization with the beta2/beta3-subunits of the GABAA receptor at the plasma membrane of granule cell somata and at cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of Purkinje cell somata. Within the molecular layer, KCC2 exhibited a distinctly punctate distribution along dendrites, indicating it may be highly localized at inhibitory synapses along these processes. The distinct postsynaptic localization of KCC2 and its colocalization with GABAA receptor in the cerebellum are consistent with the putative role of KCC2 in neuronal Cl- extrusion and postsynaptic inhibition.
Subject(s)
Antibodies/immunology , Carrier Proteins/metabolism , Symporters , Animals , Antibodies/isolation & purification , Carrier Proteins/immunology , Cell Line , Cerebellum/cytology , Cerebellum/metabolism , Chick Embryo , Culture Techniques , Humans , Immunohistochemistry , Mice , Microscopy, Fluorescence , Neurons/metabolism , Rats , Receptors, GABA-A/metabolism , Retina/cytology , Retina/metabolism , K Cl- CotransportersABSTRACT
Astrocytes play a critical role in the development of the CNS and its response to injury and disease. A key indicator of astrocyte activation is the increased accumulation of intermediate filaments composed of glial fibrillary acidic protein (GFAP). Treatment of astrocytes in vitro with transforming growth factor-beta1 (TGF-beta1) produced little morphological change, but resulted in a significant increase in GFAP mRNA and protein. Treatment with basic fibroblast growth factor (FGF-2) produced a dramatic change from a polygonal to a stellate morphology, and resulted in a significant decrease in GFAP mRNA and protein. FGF-2 also inhibited the TGF-beta1-mediated increase in GFAP mRNA and protein. Cycloheximide did not block the effects of TGF-beta1 or FGF-2 on GFAP mRNA levels, but blocked the inhibitory effects of FGF-2 on the TGF-beta1-mediated increase in GFAP expression. All effects of FGF-2 were blocked by co-incubation with 5'-methylthioadenosine, a specific inhibitor of FGF-2-induced tyrosine kinase activity and FGF receptor (FGFR) autophosphorylation. We also examined astrocyte expression of FGFR, and demonstrate the presence of FGFR 1 and 2, and lower levels of FGFR 3. Our results demonstrate that TGF-beta1 and FGF-2 cause differential effects on the astrocyte cytoskeleton and morphology, suggesting an uncoupling of process outgrowth from GFAP synthesis.
Subject(s)
Astrocytes/metabolism , Fibroblast Growth Factor 2/pharmacology , Glial Fibrillary Acidic Protein/biosynthesis , Transforming Growth Factor beta/pharmacology , Animals , Astrocytes/drug effects , Blotting, Northern , Blotting, Western , Cells, Cultured , Cross-Linking Reagents , Cycloheximide/pharmacology , Deoxyadenosines/pharmacology , Immunohistochemistry , Iodine Radioisotopes , Precipitin Tests , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/isolation & purification , Rats , Rats, Sprague-Dawley , Thionucleosides/pharmacologyABSTRACT
Traumatic injuries to the central nervous system result in astrogliosis and the formation of a dense scar at the site of the wound. Basic fibroblast growth factor (bFGF) has mitogenic and morphogenic effects on astrocytes, and an interaction between bFGF and its receptor is likely to play a role in astrogliosis. We examined trauma-induced changes in the spatial and temporal expression of FGF receptor (FGFR) in adult rats over a 28-day period following a stereotaxic lesion through the cortex and hippocampus. Immunohistochemistry and image analysis were used to evaluate the changes. Antibody characterization studies strongly suggested that staining represented FGFR 1, but did not rule out possible cross-reactivity with FGFR 2 or 3. Double immunohistochemistry for FGFR and glial fibrillary acidic protein demonstrated that mature astrocytes expressed FGFR. Expression was increased on astrocytes adjacent to the wound cavity by Day 2 postlesion. Staining increased further through Day 10 and decreased to control values by Day 28, except for a sustained increase in staining of reactive astrocytes immediately adjacent to the wound cavity. Basic FGF was detected in the nuclei of cells staining for FGFR, suggesting that FGFR-expressing astrocytes also contained bFGF. These data demonstrate a time course for astrocyte expression of FGFR that precedes and parallels the time course for astrocyte hypertrophy. Our observations suggest that endogenous bFGF, acting directly on FGFR-expressing astrocytes, may contribute to astrogliosis.
