| Glias are traditionally thought to assume a structural, trophic, and protective role to neurons. With the development of research techniques and the further study on glias, mounting evidences have suggested the bidirectional signaling between neurons and glias. Glias may be an integral part of the communication network within the central nervous system and regulate neuronal activity. It is known that astrocytes take a key role in the response to hypoosmotic stimulation: astrocytes can perceive the change of osmotic pressure and release taurine which then activates glycine receptors on the neurons, and inhibits the release of VP from the neurons in SON. While it is not clear that whether SON astrocytes respond to hyperosmotic stimuli and what's the relationship between them and neurons?Based on the previous researches, the present study investigated the response of SON neurons and glias (astrocytes and microglias) to hyperosmotic stimulation and their relationship to find out the roles of glias in regulating hyperosmotic stimulation.Immunohistochemistry method was used to observe the temporal and spatialexpression of NMDAR2, signal molecules, skeleton proteins and connexins in SON neurons and glias (astrocytes and microglias). Radioimmunoassay was used to detect vasopressin (VP) content in plasma before and after hyperosmotic stimulation.Ultrastructure between activated SON astrocytes and neurons was observed by double immune-electron-microscopic staining method.We injected carbennoxolone, a gap junction blocker, into the lateral ventricle, which was followed by hyperosmotic stimulation, the immunohistochemical staining of neurons and astrocytes and VP content in plasm were studied.Western blot was performed to detect the content of Cx43 and Cx32 in SON following hyperosmotic stimulation.Treat primary cultured neurons and astrocytes with hyperosmotic stimulation. Immunofluorescence was used to study the expression of Cx43 and Cx32 in SON neurons and astrocytes. By means of Fluo-3/AM and laser scanning confocal microscopy, we also observed intracellular calcium transient in astrocytes and microglias.The main results included:1, After hyperosmotic stimulation, there emerged in rat SON the activated astrocytes which showed NMDAR2, Fos/GFAP and Cx43 positive immuno-histochemical staining, the activated neurons which showed PLC, NMDAR2, Fos and Cx32 staining, and also OX42 positive stained microglias. The activated astrocytes and microglias emerged earlier than the activated neurons. These activated cells formed intimate temporal and spatial relationship.2, The electron dense area (EDA) consisting of the astrocytic process on one side and the neuron (dendrite) on the other side was observed in immune-electron-microscopic staining studies, and the EDA was characterized withdouble layers thickening and dark staining cytomembranes with a narrow cleft between them. Cx32-LI appeared on the neuron side or and Cx43-LI on the astrocyte side respectively. This structure increased obviously following hyperosmotic stimulation.3, VP content in plasma increased significantly 45min after hyperosmotic stimulation. When pre-injected carbennoxolone, a gap junction blocker, into the lateral ventricle, followed by hyperosmotic stimulation, VP content remained the base line. The expression of GFAP-LI astrocytes showed no difference, while that of Fos-LI neurons decreased significantly.4, Western blot showed increase of Cx43 and Cx32 in SON and also a translocation of them from plasm to membrane. After hyperosmotic treatment, Cx43-LI granules increased quickly on the plasm of the cultured astrocytes and Cx32-LI increased apparently in the cultured neurons.5, After being treated with hyperosmotic stimulation, the cultured astrocytes and microglias showed a fast increase of intracellular calcium concentration followed by a decrease. The [Ca2+]j of astrocytes decreased slower than that of microglia...
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