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Hypotonic stimulation can cause the supraoptic nucleus astrocytes synthesis and release of the inhibitory neurotransmitter taurine, thereby inhibiting the synthesis and release of VP of neurons. Known brain exists Glutamate the mediated astrocyte-neuron signaling. SON is one of the brain osmoregulation hub exists within the SON Glutamate the mediated astrocyte-neuron signaling whether to play a regulatory role in hypertonic stimulation, its mechanism of action? Were three groups for this study to explore this issue (Experiment I, II and III). In addition, acute and chronic hypertonic stimulation of glial cells different? Our research (experimental). Reaction the experimental acute intravenous hypertonic stimulation SON neurons depends on the activity of the purpose of the AST: acute intravenous hypertonic (HS) stimulus SON within the AST and the reaction of neurons and their relationship. Methods: 65 SD rats were divided into four groups: the first group of normal control group (n = 5) without any processing; 2 isotonic stimulation group (IS, n = 20), the isotonic saline ( 0.9% NaCl) were injected into the tail vein; Group 3 for the the hypertonic stimulation group (HS, n = 20), hypertonic saline (9% NaCl) was injected into the tail vein; Group 4 the fluorocitrate HS group (FCA plus HS group, n = 20), advance to the lateral ventricle injected slowly 1nmol / 1 m FCA h after hypertonic stimulation; more than animals, apart from the first group, the other groups were, respectively, survival 15,45,90 and 180 5 min, at each time point. Conventional perfusion fixation, slices containing the SON slices anti VP, anti-Fos, anti-GFAP single or double immunofluorescence staining. HS stimulation before and after stimulation 45min, extracting the rats in each group venous blood by radioimmunoassay determination of plasma VP concentration. Results: 1. Acute intravenous HS stimulation SON within AST is activated, the obvious response, manifested as larger cell body processes thicker, GFAP deeply stained, mean fluorescence intensity, thickening of the SON-VGL thickness, Fos / GFAP double The labeled cells were increased above reaction peaked 45min after stimulation. Stimulation in SON neurons is activated, Fos-positive nuclei significantly increased the number of VP-positive neurons increased significantly, both peak are 90min. 3.FCA can significantly interfere with the reaction between the AST and neurons. 4. Stimulated plasma VP level was significantly elevated, FCA pre-processing to give HS stimulate plasma VP level rise. Conclusion: Intravenous HS after stimulation in SON AST sensitive peaked 45min after stimulation, Fos and VP neurons 90min, plasma VP concentration also increased expression of the peak are stimulated. FCA AST metabolic inhibitor, the above AST and neuronal responses were blocking; This indicates that the reaction the SON neurons HS stimulation depends on AST activity. AST release via Cx43 hemichannel stimulation experiment two intravenous HS Glu regulate neuronal activity purposes: to explore the HS after stimulation in SON AST adjustment mechanism for neurons. Method: 1. Overall animal experiments: 25 male SD rats were divided into five groups (5 mice / group): Group 1 was the control group; 2 IS group; Group 3 HS group; 4th group FCA plus HS group; Group 5 as carbenoxolone HS group (CBX plus HS group); Group 6 the PPADS HS group; Group 7 the BBG HS group. To production animal model experiment, all animals survived 45min conventional fixed slice, slice the anti of NMDAR-2 anti-glutamate or anti of GFAP, anti of Cx43, single or double immunofluorescence staining of anti-Fos, anti-VP. Cultured cells: the use of two kinds of cell model: (1) primary cultured AST, take the 18-day fetal rat hypothalamus, the conventional separation of purified culture AST 2 weeks, the cultured cells were divided into six groups: Group 1 IS group (Ca2 / Mg2-free DMEM culture); Group 2 HS group; Group 3 for the gap junction blocker (CBX Gap26) HS group; Group 4 for the glutamate transporter inhibitor ( Su hydroxyaspartate, THA) HS group, 5th the group P2Xn receptor antagonist (PPADS and BBG) HS group, six groups as the Ca2 chelating agents (BAPTA-AM) HS group, each group handling a 3,5,7,9,10,12,15 min. (2) C6 cell line grouping ibid. Respectively, to close the the above primary cultured AST culture medium used for HPLC detection Glu content. C6 cells was measured by flow cytometry (FCM) Glu Cx43 content results: 1. Overall animal experiments: the acute venous HS stimulation, caused SON following changes: (1) AST Glu significantly upregulated, FCA interfere with its raised, CBX is not interference. (2) AST of Cx43 expression is significantly increased, FCA interfere with its raised, CBX is not interference. (3) neurons NMDAR-2 expression was significantly increased the FCA and CBX prevent its raised. (4) increased expression of neurons within the VP, FCA, CBX inhibit the rise of the VP. Culture cell experiments (1) the HS stimulated C6 intracellular Glu expression increased significantly, 3min peaked later dropped. The primary AST culture medium the Glu level peaked in 5min, declining thereafter. (2) CBX or Gap26 HS treated C6 intracellular Glu content increased significantly, and has been maintained at high expression levels, the primary AST culture medium Glu content compared with the HS group decreased. (3) THA HS group compared with the HS group, Glu level in the culture medium showed no significant difference. (4) PPADS, or BBG HS group, compared with the HS group, the cells and culture medium Glu content showed no significant differences. (5) BAPTA-AM HS group compared with the HS group, the cells and culture medium Glu content showed no significant differences. (6) cultured cells Cx43 expression was also significantly increased. Conclusion: 1. Activated after intravenous HS in SON AST is activated, AST increased synthesis of Glu release through Cx43 hemichannels acting on SON neurons of of NMDAR-2, increase the synthesis and release of VP. Activate 2.HS cultured glial cells, increased synthesis of Glu and Cx43, the CBX or Gap26 not affect the synthesis of Glu, but can block the release; THA PPADS, BBG and BAPTA-AM Synthesis of Glu release had no significantly affected. Experimental high (low) infiltration stimulation of glial cells release neurotransmitters Objective: explore the HS or hypotonic stimulation glial cells release neurotransmitters? ATP is involved in the regulation of osmotic pressure. Method: 1. Cultured cells used in this study two kinds of glial cells cultured model: (1) primary cultured AST to take 18 days of fetal rat hypothalamus, the conventional separation and purification cultured for 2 weeks; (2) C6 cell line . All cultured cells were divided into three groups: Group 1 for the IS group; Group 2 HS group; each group are processing 1, 3, 5, 10, 15 min. The three groups for the the hypotonic stimulation group; each group are processing 1, 3, 5, 10, 15 min. 2. Respectively to close above culture medium and intracellular fluid by HPLC primary culture the AST extracellular fluid Glu, and taurine content, FCM determination C6 cells, Glu, and taurine content. 3 using fluorescein - the the luciferase enzymatic detection of primary cultured AST intracellular fluid and extracellular ATP content. In results: 1.HS stimulus promote AST main synthetic release Glu, taurine no change hypotonic stimulation to promote AST synthesis and release of taurine, Glu no change 3. Compared with isotonic osmotic changes, was no significant change in the intracellular and extracellular ATP levels. Conclusion: HS stimulus caused AST main synthetic release Glu excitatory neural element release VP. Hypotonic stimulation to promote AST synthesis and release of taurine, VP inhibitory neurons release. ATP was not involved in the adjustment process. Experimental acute and chronic HS stimulate neurons in SON AST differences Objective: To investigate the acute, chronic the HS stimulus of rat behavior in SON AST and neurons differences. Method: 1. Animal groups: 25 male SD rats were divided into five groups (5 / group): Group 1 was the control group; Group 2 2% hypertonic saline feeding 3d group; Group 3 2% hypertonic saline feeding 6d group; Group 4 2% the hypertonic saline feeding 9d group; the 5 abdominal cavity 9% hypertonic saline stimulation group, the animals survived 45min. General detection: Determination of body weight of rats eating. Determination of mechanical pain threshold of rats and nerve function. Immune glory staining all animals require time point of death, anti-conventional fixed slice, slice of GFAP, anti VP, anti-Fos, anti-Cx43 single the dual epidemic staining. Results: 1. Acute intraperitoneal hypertonic SON AST intracellular GFAP and Cx43 fluorescence intensity significantly increased 45min after stimulation, the labeled SON-VGL thickness of GFAP marker Cx43 increase in the number of Fos / GFAP double-labeled cells, while AST's growth projections, thicker, larger cell bodies, was activated AST form. In addition, the number of VP and Fos positive neurons also increased significantly. 2. 2% hypertonic saline group (1) fed hypertonic saline 3d, compared with the control group, Cx43 and GFAP expression was significantly increased, the the labeled SON-VGL thickness of GFAP marker Cx43 also increased, but less than The acute intraperitoneal hypertonic stimulation group. Fos / GFAP double the number of labeled cells are also a significant increase in the number of VP and Fos positive neurons was significantly increased. AST protrusions retracted, the cell size is no change. (2) Hello hypertonic saline 6d, Cx43, GFAP expression decreased the the labeled SON-VGL thickness of GFAP-labeled with Cx43 significantly reduce the number of the Fos / of GFAP double the number of labeled cells as well as VP and Fos positive neurons of also significantly reduced, but still higher than normal. (3) Hello hypertonic saline 9d AST projections retraction, cell body atrophy, Cx43, GFAP expression significantly decreased number of of Fos / GFAP double the number of labeled cells, VP and Fos positive neurons were also significantly reduced, GFAP marker the marked with Cx43 SON-VGL thickness were significantly reduced. Mechanical pain threshold (4) acute intraperitoneal hypertonic stimulus 45min, rats, weighing no significant change, while the feed brine 3d mechanical pain threshold rats gradually increased, lighter weight, reduced food intake to 9d peak prompt physical function in rats in a failure state. Ming camera obscura determination of nerve function in rats found that the reduced ability of learning and memory in rats. Conclusion: acute and chronic hypertonic stimulation of AST differ, the acute stimulation can activate AST, and thus participate in modulating neuronal activity, and chronic hypertonic after stimulation, significantly enhanced the activity in the early (3d) of AST and neural element to late reaction (6,9 d) bodily functions in failure, the reaction function is significantly diminished in rats, AST and neurons also significantly inhibited.
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