We studied the effects of water deprivation on the phosphorylation of TrkB and NMDA receptor subunits in the supraoptic nucleus (SON) of the rat. Laser capture microdissection and qRT-PCR was used to demonstrate BDNF and TrkB gene expression in vasopressin SON neurones. Immunohistochemistry confirmed BDNF staining in vasopressin neurones, while staining for phosphorylated TrkB was increased following water deprivation. Western Blot analysis of brain punches containing the SON revealed that tyrosine phosphorylation of TrkB (pTrkBY515), serine phosphorylation of NR1 (pNR1S866 or pNR1) and tyrosine phosphorylation of NR2B subunits (pNR2BY1472 or pNR2B) were significantly increased in WD animals compared to control. Access to water for 2 h reduced pTrkBY515 content to control levels without affecting pNR1 or pNR2B. Four hours of rehydration was needed to reduce pNR1 and pNR2B to control. To test whether increased phosphorylation of TrkB in this study is mediated by BDNF, a group of animals were instrumented with right SON cannula coupled to mini-osmotic pumps filled with vehicle or TrkB-Fc fusion protein which prevents BDNF binding to TrkB. In the left SON contralateral to the cannula, TrkB phosphorylation was significantly enhanced following WD. Separate analysis of the right SON, which received TrkB-Fc, showed that the TrkB receptor phosphorylation following WD was significantly attenuated. While increased pNR1S866 following WD was not affected by local infusion of TrkB-Fc, pNR2BY1472 was significantly reduced. Co-immunoprecipitation revealed an increased physical interaction between Fyn kinase and NR2B and TrkB in the SON following water deprivation. Thus, activation of TrkB in the SON following WD may affect cellular excitability through the phosphorylation of NR2B subunits.
The purpose of this study was to measure the expression of transient receptor potential (TRP) channels in the magnocellular neurons of the paraventricular (PVN) and supraoptic nucleus (SON) in an animal model of hepatic cirrhosis associated with inappropriate vasopressin (AVP) release. In these studies we used chronic bile duct ligation (BDL) in the rat, a commonly used model of hepatic cirrhosis, associated with elevated plasma AVP. This study tested the hypothesis that changes in TRPV channel expression may be related to inappropriate AVP release in BDL rats. To test our hypothesis, we utilized laser capture microdissection of AVP neurons in the PVN and SON and Western blot analysis from brain punches. Laser capture microdissection and qRT-PCR demonstrated elevated TRPV2 mRNA in the PVN and SON of BDL as compared to sham ligated controls. AVP transcription was also increased as determined using intron specific primers to measure heteronuclear RNA. Immunohistochemistry demonstrated increased AVP and TRPV2 positive cells in both the PVN and SON after BDL. Also, there was an increased co-expression of TRPV2 and AVP cells after BDL. However, there was no change in the colocalization counts of TRPV2 and OXY in both the magnocellular regions evaluated. In the SON but not the PVN, transcription levels of TRPV4 was also significantly increased in BDL rats Western Blot analysis of punches containing the PVN and SON revealed that TRPV2 protein content was significantly increased in these brain regions in BDL rats compared to sham. Our data suggests that regionally specific changes in TRPV expression in the MNC AVP neurons could alter their osmosensing ability.
Bile duct ligation (BDL), a model of hepatic cirrhosis, is associated with dilutional hyponatremia and inappropriate vasopressin release. ΔFosB staining was significantly increased in vasopressin and oxytocin magnocellular neurosecretory cells in the supraoptic nucleus (SON) of BDL rats. We tested the role of SON ΔFosB in fluid retention following BDL by injecting the SON (n = 10) with 400 nl of an adeno-associated virus (AAV) vector expressing ΔJunD (a dominant negative construct for ΔFosB) plus green fluorescent protein (GFP) (AAV-GFP-ΔJunD). Controls were either noninjected or injected with an AAV vector expressing only GFP. Three weeks after BDL or sham ligation surgery, rats were individually housed in metabolism cages for 1 wk. Average daily water intake was significantly elevated in all BDL rats compared with sham ligated controls. Average daily urine output was significantly greater in AAV-GFP-ΔJunD-treated BDL rats compared with all other groups. Daily average urine sodium concentration was significantly lower in AAV-GFP-ΔJunD-treated BDL rats than the other groups, although average daily sodium excretion was not different among the groups. SON expression of ΔJunD produced a diuresis in BDL rats that may be related to decreased circulating levels of vasopressin or oxytocin. These findings support the view that ΔFosB expression in SON magnocellular secretory cells contribute to dilutional hyponatremia in BDL rats.
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