The Responses And Relationship Of Astrocytes And Neurons In The Supraoptic Nucleus After Hypertonic Stimulation

It is well known that the hypotonic stimulus induce the synthesis and release of taurine from astrocytes which inhibit the synthesis and release of VP. In brain, glutamate mediated astrocyte-neuron signaling and SON is the center of osmotic regulation. Is the glutamate mediated astrocyte-neuron signaling included in the regulation of SON under hypertonic stimulus. In order to clarify this question, we performed three experiments (first, second and third experiments). Moreover, is there difference between the acute and chronic hypertonic stimulus on the astrocytes? The fourth experiment was performed.The first experimentAcute intravenous hyperosmotic stimulus induces activation in the neurons depends on the regulation of astrocytes in supraoptic nucleus Objective To investigate the response and relationship between the astrocytes and neurons in SON induced by acute intravenous hypertonic stimulus.Methods 65 SD rats were divided into four groups: control group(n=5), Iso-osmia group(IS group,n=20), hypertonic stimulus group(HS group,n=20) and FCA plus HS group(n=20). Except the control group, the rats in other three groups were survived for 15, 45, 90 and 180 min,n=5/per time point, fixation, cut section and anti-VP, anti-Fos, anti-GFAP immunofluorescent staining were preformed by usual methods.We also detected the VP content in plasma by radioimmuno assay. Assays were performed blindly.Result 1.HS 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 stimulus, the response peaked.2.HS also actived the neurons in SON. The expression of Fos and VP positive neurons were increased.The responsed peak was 45min.3.The FCA inhibited the response of astrocytes and neurons after HS.4.VP level in plasma was increased after HS and this effect could be inhibited by FCA.Conclusion Intravenous hypertonic stimulus induced the response of astrocyte and neuron. This effect could be inhibited by FCA----metabolic inhibitor. It is suggested that the activation of neurons should depend on the the activation astrocytes after intravenous HS in SON.The second experiment Astrocytes regulate the activation of neurons by release glutamate through Cx43 hemichannel in SON after the intravenous hypertonic stimulusObjective To investigate mechanism of astrocytes regulation neurons in SON.Methods 1.35 SD male rats were divided into 7 groups(5 rats/per group): control group, IS group, HS group, FCA plus HS group,CBX plus HS group, PPADS plus HS group and BBG plus HS group. All rats were survived for 45min, fixation, cut section and anti- NMDAR-2, anti-glutamate, anti-GFAP, anti-Cx43, anti-Fos and anti-VP immunofluorescent staining were performed by usual methods.2. Cultured cells (1)Primary cultured astrocytes:Cultured astrocytes were divided into 6 groups: IS group, HS group, Gap junction inhibitor(CBX and Gap26) plus HS group, THA plus HS group, P2XnR antagonist(PPADS and BBG) plus HS group and Ca2+chelator(BAPTA-AM) plus HS group.Every group was treated by HS for 1,3,5,7,9,10,12,15min. (2)C6 cells:We cultured C6 cells and divided it into 6 groups as above.Culture fluid of primary cultured astrocyte was collected for detecting glutamate content by HPLC. Intracelluar glutamate content of C6 cell was determined by flow cytometry(FCM).Results 1.In vivo, FCA inhibited the increase of glutamate and Cx43 after HS while CBX had no the effect. At the same time, the expression of NMDAR-2 and VP were blocked by FCA and CBX.2.In vitro, the C6 intracelluar glutamate content or extracelluar fluid glutmamte of cultured astrocyte was increased and peaked at 3min or 5min respectively after HS. After treated by CBX or Gap26, the C6 intracelluar glutamate content kept the high expression. However, the glutamate content in extracelluar fluid was decreased. Moreover,THA, PPADS, BBG or BAPTA-AM has no effect on the intracellular or extracelluar glutamate content. HS also induced the expression of Cx43.Conclusion Intravenous HS induced the synthesis and release of glutamate from astrocyte through Cx43 hemichannel. And then the glutamate affected the NMDAR-2 and increased the synthesis and release of VP.The third experiment The effect of hypertonic(hypotonic) stimulus on the transmitter release from astrocytesObjective To investigate if the hypertonic or hypotonic stimulus induced the astrocyte to release different transmitter and ATP was included in the regulation of osmotic pressure.Methods 1.Cultured cells: Primary cultured astrocytes and C6 cells were divided into three groups: IS group, HS group and hypotonic stimulus group. HS group and hypotonic stimulus groups were both treated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.and 15 min.2.Culture fluid of primary cultured astrocytes was collected for detecting glutamate and taurine content by HPLC. Intracelluar glutamate and taurine content of C6 cells were determined by FCM.3.Luciferin-luciferase based luminescence assay was used for detecting the ATP content.Results 1.HS induced the release of glutamate(not taurine)from astrocyte2.Hypotonic stimulus induced the release of taurine(not glutamate) from astrocyte.3.ATP had no change with the change of osmotic pressure. Conclusion HS induced the release of glutamate from astrocytes and activated the neurons to release VP. Hypotonic stimulus induced the release of taurine from astrocytes which inhibited the release of VP from neurons. ATP was not included in the regulation of osmotic pressure in SON.The fourth experiment The difference between the effects of acute and chronic hypertonic stimulus on the astrocytes and neurons in SONObjective To investigate the different between the acute and chronic HS on the astrocytes and neurons in SONMethods 1.25 SD male rats were divided into 5 groups(5 rats/group): control group, HS 3d group(feeding 2% hypertonic saline for 3d), HS 6d group(feeding 2% hypertonic saline for 6d), HS 9d group(feeding 2% hypertonic saline for 9d), acute HS group.2.Determined body weigh and foodintake.3.Detected the paw withdrawal mechanical threshold.4.The anti-GFAP, anti-Cx43, anti-Fos and anti-VP immunofluore-scent staining were preformed by usual methods.Results 1. At 45min after acute celiac HS, SON astrocytes 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 and VP/Fos positive neurons significantly increased.2. Feeding 2% hypertonic saline group:(1)After feeding hypertonic 2% saline for 3d, compared with control group, the expression of Cx43 and GFAP were increased and the thickness of SON-VGL thickened.And at the same time, the Fos/GFAP double labeled astrocytes and VP/Fos positive neurons also increased. However, the changes were fewer than that in the acute hypertonic stimulus.(2)After feeding hypertonic 2% saline for 6d, the expression of Cx43 and GFAP, the thickness of SON-VGL, and the number of VP/Fos positive neurons all decreased compared with acute HS group.(3)After feeding hypertonic 2% saline for 9d, the expression of Cx43 and GFAP, the thickness of SON-VGL, and the number of VP/Fos positive neurons all obviously decreased compared with acute HS group. And the cell body exhibited atrophy with the retracted processes.(4)For the acute HS group, the paw withdrawal mechanical threshold and body weigh had no change. However, after feeding 2% saline for 3d, the paw withdrawal mechanical threshold was higher than that in the acute HS group. The body weigh and foodintake were also decreased. By the detecting of ethology, the ability of learning and memory also decreased.Conclusion Acute and chronic HS have different effect on the astrocytes.Acute HS could activate astrocytes which regulate the neurons in turn. However, after the chronic HS, the astrocytes and neurons were activated at the early stage(3d) and inhibited at the later stage(6d, 9d).The phenomenom prompted us that the rats were exhausted at the later stage of chronic HS which inhibited the astrocytes and neurons.