The Mechanisms Of Astrocytes In The Supraoptic Nucleus Regulating The Activation Of Neurons Induced By Acute Peritoneal Hypertonic Stimulation

To study a route of signal from the peripheral osmotic receptors reaches the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and the response and relationship each other of the astrocytes and neurons in the dorsal horn of spinal cord (DHSC), nucleus of tract solitarius (NTS) and SON to acute hypertonic stimulation via peritoneal , and the role and mechanism of astrocytes regulating the activation of neurons induced by hypertonic stimulation. The flowing four experiments were performed.The first experiment The peripheral and central neural axonal terminals directly termite on the astrocytesObjective To investigate whether the axonal terminals form peripheral or central nerve directly end on the astrocytes.Methods Ten SD rats were divided into two groups: The rats of first group were performed cut left vagal trunk in the neck under an operating microscope and survived for 30 days. The tissues from the brain and spinal cord of experimental animals were performed according to immunoelectro microscopic methods. The rats in second group, 10 % Biotinylated dextran amine (BDA, Vector, diluted with 0.9 % NaCl, 0.2μl/per rat) was injected into right NTS (n=5) through microsyringe (0.47 mm in diameter) over a period of 10 min, as BDA anterograde tracing model, after 5 days were fixed, cut sections and performed immunofluorescent staining and observed under confocal microscope.Results (1) The typical neuronal synapses (including symmetrical and asymmetrical synapses) and synaptoid contacts formed by axonal terminals and GFAP positive astrocytic processes in NTS were found. (2) Some (64%) of the degenerate axonal terminals induced vagotomy directly terminate on the neurons, or astrocytes labeled with GFAP, or both neurons and astrocytes and formed a synaptoid contacts. (2) BDA injection area located in right NTS, which revealed many BDA labeled neurons and dense fibers. In SON 40 % BDA labeled fibers directly contacted with VP positive neurons, and 36 % BDA positive fibers contacted with astrocytes labeled by GFAP. Immunoeletromicroscope observed that the axonal terminals directly contacted with GFAP positive processes and formed synaptoid contacts.Conclusion the astrocytes directly contacted with afferent fibers from peripheral or central neural system and formed a synaptoid contacts. The second experiment The route from the peripheral osmoreceptors reaching supraoptic nucleus involved in regulating osmolarityObjective To research the neural route of transmitting osmotic signal from the peripheral osmotic receptors to SON and PVN, and the response of astrocytes and neurons in interrelated areas to acute hypertonic stimulation. Methods Thirty SD rats were divided into six groups:①The rats in the first group did not receive any treatment, as a normal control model.②The second group was isotonic saline (IS) model, 4 ml isotonic saline (0.15 mol/L) was injected into peritoneaus.③The third group was hypertonic saline (HS) model, 4 ml hypertonic saline (1.54 mol/L) was injected into the peritoneaus, these animals survived for 45 min.④The fourth group was bilateral subdiaphragmatic vagotomy (SDV) plus HS model.⑤The fifth group was bilateral splanchnic nerve lesion (SNL) plus HS.⑥The sixth group was sham-operated plus HS. 7 days after SDV, SNL, or sham-operared, these rats were gave HS and survived 45 min. The sections from dorsal horn of spinal cord (DHSC), or NTS of medullary oblongata, or SON were preformed immunofluorescent staining.Results (1) The clear response of astrocytes and neurons in DHSC of above mentioned experimental animals did not observe. (2) HS stimulation induced Fos positive neurons and GFAP positive astrocytes significantly increased in NTS and the area postrema (AP), and revealed marked difference as compared with IS group. SDV blocked the response, but SNL can not block. (3) HS induced that the astrocytes were activated and GFAP expression increased and the mean fluorescent intensity of GFAP intensified, the mean thickness of SON-VGL thickened, the mean number of Fos/GFAP double labeled astrocytes significantly increased. These response was blocked by SDV, but SNL can not block. Conclusion The signal from peripheral osmotic receptors transmitted to NTS, AP via vagus, the fibers from NTS projected to SON. The splanchnic nerve did not involve in osmotic signal transmitting. The NTS and AP play important role in osmotic signal transmitting, but DHSC did not play role.The third experiment Acute hyperosmotic stimulus induces activation in the neurons depends on the regulation of astrocytes in supraoptic nucleusObjective To investigate the response and relationship each other of the astrocytes and neurons in SON induced by acute hypertonic stimulation.Methods Eighty-five rats were divided into five groups: The first group was normal control group (n=5). The second was IS group (n-20). The third group was HS group (n-20). The fourth group, a glial metabolic inhibitor, fluorocitrate (FCA, 1μl /rat, 1nmol/μl, W/V, n=20) and the fifth group, a gap junction blocker, carbenoxolone (CBX, 30μg/rat, 10μg/μl, W/V, n=20) was pre-injected into lateral ventricle respectively followed by HS 2 h later as FCA or CBX plus HS models. All experimental animals, ecepted normal contol group, were survived for 15, 45 90 and 180 min respectively, n=5/per time point, fixation, cut section and anti-Fos, anti-GFAP immunofluorescent staining were preformed by usual methods.Results (1) Hypertonic stimulation induced SON astrocytes clearly responding and revealed marked cellular hypertrophy with thickened processes, the mean fluorescent intensity of GFAP staining intensified, the mean thickness of SON-VGL thickened, the mean number of Fos/GFAP double labeled astrocytes significantly increased. At 45 min after stimulation the response peaked. (2) Fos positive neurons significantly increased and peaked at 90 min after stimulation. (3) FCA markedly blocked the response both astrocytes and neurons, while CBX only inhibited neuronal response, was not astrocytes.Conclusion Hypertonic stimulation induced the response of SON astrocytes was early than neurons. HS induces Fos expression in SON neurons depends on the activation of astrocytes.The fourth experiment The glutamate released from astrocytes via connexin 43 hemichannels is required for regulating hypertonicity induced activation of the supraoptic neuronsObjective To research mechanisms of SON astrocytes regulating the activation of neurons induced by hypertonic stimulation.Methods (1) For immunofluorescent staining, twenty-five SD rats were divided into five groups (5 rats/per group): the first group was normal control group, the second group was IS group, the third group was HS group, the fourth group was FCA plus HS group and the fifth group wa CBX plus HS group. All animals were survived for 45 min, fixation, cut section and anti-glutamate, anti-NMDAR-2, anti-VP immunofluorescent staining were preformed by usual methods. (2) Cultured astrocytes from the hypothalamus of rat embryos (E18) were prepared and maintained in culture as described previously (Ye et al., 2003). Astrocytes were plated on 60-mm Primaria plastic culture dishes (Nunc Clone) with or without glass coverslips (1×105 cells/cm2 24 h before the experiment) and kept at 37oC in a 5% CO2/95% air atmosphere at nearly 100% relative humidity. Cultures were maintained in growth medium (Dulbecco's modified Eagle's medium, DMEM) and reached confluence within 2 weeks. All cultured cells were divided into four groups: the first group was IS DMEM group, the second group was group treated by HS DMEM for 1, 3, 5, 10 and 15 min, the third group was pre-treated by CBX for 30 min next moved to IS DMEM, the fourth group was pre-treated by CBX for 30 min next moved to HS DMEM for 1, 3, 5, 10 and 15 min. These cells were fixed and anti-glutamate and anti-GFAP double immunofluorescent staining. (3) Glutamate and taurine measurement, using HPLC measured concentration of glutamate and taurine in astrocytes medium. (4) For immunoelectro-microscope ten rats were divide into IS group and HS group, anti-connexin 43 (Cx43) and anti-NMDAR 2 double immunoelectromicroscopic staining was performed. (5) Plasma VP content radioimmunoassay. The blood collected from the femoral veins of rats as described above, was centrifuged at 1500×g for 15 min at 4oC. The plasma was separated and stored as aliquots in plastic tubes at–70oC until used. All samples were measured using the vasopressin 125I RIA kit (DiaSorin Company). Assays were performed blindly.Results (1) Hypertonic stimulation significantly increased glutamate expression in SON strocytes, FCA markedly inhibited both glutamate and GFAP signals in astrocytes, but CBX did not inhibit. (2) Hypertonic stimulation induced the expression of NMDAR 2 in neurons markedly up-regulated, FCA and CBX inhibited its up-regulation. (3) Hypertonic stimulation increased the expression of VP in SON neurons, FCA, CBX and SDV inhibited its increasing, but SNL can not inhibit. (4) Hypertonic stimulation induced the expression of glutamate and GFAP in cultured astrocytes significantly up-regulated, at 3min peaked. The cells pre-treated by CBX, hypertonic stimulation increased glutamate expression and maintained high level. (5) Hypertonic stimulation significantly increased the concentration of glutamate in medium, but the concentration of taurine did not increase. (6) After hypertonic stimulation, the number of Cx43 hemichannels in SON significantly increased.Conclusion Hypertonic stimulation activated SON astrocytes, activated astrocytes synthesized glutamate and released it via Cx43 hemichannels, glutamate signal activated neurons via NMDAR and increased synthesis and release of VP.