Joseph D. Walch

Brain-Derived Neurotrophic Factor-Tyrosine Kinase B Pathway Mediates NMDA Receptor NR2B Subunit Phosphorylation in the Supraoptic Nuclei Following Progressive Dehydration

We studied the effects of water deprivation (WD) on the phosphorylation of tyrosine kinase B (TrkB) and NMDA receptor subunits in the supraoptic nucleus (SON) of the rat. Laser capture microdissection and quantitative reverse transcriptase polymerase chain reaction was used to demonstrate brain‐derived neurotrophic factor (BDNF) and TrkB gene expression in vasopressin SON neurones. Immunohistochemistry confirmed BDNF staining in vasopressin neurones, whereas staining for phosphorylated TrkB was increased following WD. 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 controls. 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 levels. To test whether increased phosphorylation of TrkB in the present 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. Although 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 WD. Thus, activation of TrkB in the SON following WD may affect cellular excitability through the phosphorylation of NR2B subunits.

ΔFosB in the supraoptic nucleus contributes to hyponatremia in rats with cirrhosis

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.

BDNF-TrkB Pathway Mediates NMDA receptor NR2B Subunit Phosphorylation in the Supraoptic Nuclei Following Progressive Dehydration

We studied the effects of water deprivation (WD) on the phosphorylation of tyrosine kinase B (TrkB) and NMDA receptor subunits in the supraoptic nucleus (SON) of the rat. Laser capture microdissection and quantitative reverse transcriptase polymerase chain reaction was used to demonstrate brain‐derived neurotrophic factor (BDNF) and TrkB gene expression in vasopressin SON neurones. Immunohistochemistry confirmed BDNF staining in vasopressin neurones, whereas staining for phosphorylated TrkB was increased following WD. 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 controls. 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 levels. To test whether increased phosphorylation of TrkB in the present 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. Although 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 WD. Thus, activation of TrkB in the SON following WD may affect cellular excitability through the phosphorylation of NR2B subunits.

Region specific changes in TRPV channel expression in the vasopressin magnocellular system in hepatic cirrhosis induced hyponatremia

The present study aimed to measure the expression of transient receptor potential (TRP) channels in the magnocellular neurones 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, which is a commonly used model of hepatic cirrhosis, associated with elevated plasma AVP. The present study tested the hypothesis that changes in TRP vanilloid (TRPV) channel expression may be related to inappropriate AVP release in BDL rats. To test our hypothesis, we utilised laser capture microdissection of AVP neurones in the PVN and SON and western blot analysis from brain punches. Laser capture microdissection and quantitative reverse transcriptase‐polymerase chain reaction demonstrated elevated TRPV2 mRNA in the PVN and SON of BDL 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 colocalisation counts of TRPV2 and oxytocin in both the magnocellular regions evaluated. In the SON but not the PVN, the transcription levels of TRPV4 were 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 rats. Our data suggest that regionally specific changes in TRPV expression in the magnocellular neurosecretory cell AVP neurones could alter their osmosensing ability.

Central Control of Fluid and Electrolyte Homeostasis: ANG II receptor subtype 1a gene knockdown in the subfornical organ prevents increased drinking behavior in bile duct-ligated rats

Bile duct ligation (BDL) causes congestive liver failure that initiates hemodynamic changes, resulting in dilutional hyponatremia due to increased water intake and vasopressin release. This project tested the hypothesis that angiotensin signaling at the subfornical organ (SFO) augments drinking behavior in BDL rats. A genetically modified adeno-associated virus containing short hairpin RNA (shRNA) for ANG II receptor subtype 1a (AT1aR) gene was microinjected into the SFO of rats to knock down expression. Two weeks later, BDL or sham surgery was performed. Rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. The rats were euthanized 28 days after BDL surgery for analysis. A group of rats was perfused for immunohistochemistry, and a second group was used for laser-capture microdissection for analysis of SFO AT1aR gene expression. BDL rats showed increased water intake that was attenuated in rats that received SFO microinjection of AT1aR shRNA. Among BDL rats treated with scrambled (control) and AT1aR shRNA, we observed an increased number of vasopressin-positive cells in the supraoptic nucleus that colocalized with ΔFosB staining, suggesting increased vasopressin release in both groups. These results indicate that angiotensin signaling through the SFO contributes to increased water intake, but not dilutional hyponatremia, during congestive liver failure.

ANGIOTENSIN II RECEPTOR SUBTYPE 1A (AT1AR) GENE KNOCKDOWN IN THE SUBFORNICAL ORGAN (SFO) PREVENTS INCREASED DRINKING BEHAVIOR IN BILE DUCT LIGATED RATS.

Bile duct ligation (BDL) causes congestive liver failure that initiates hemodynamic changes, resulting in dilutional hyponatremia due to increased water intake and vasopressin release. This project tested the hypothesis that angiotensin signaling at the subfornical organ (SFO) augments drinking behavior in BDL rats. A genetically modified adeno-associated virus containing short hairpin RNA (shRNA) for ANG II receptor subtype 1a (AT1aR) gene was microinjected into the SFO of rats to knock down expression. Two weeks later, BDL or sham surgery was performed. Rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. The rats were euthanized 28 days after BDL surgery for analysis. A group of rats was perfused for immunohistochemistry, and a second group was used for laser-capture microdissection for analysis of SFO AT1aR gene expression. BDL rats showed increased water intake that was attenuated in rats that received SFO microinjection of AT1aR shRNA. Among BDL rats treated with scrambled (control) and AT1aR shRNA, we observed an increased number of vasopressin-positive cells in the supraoptic nucleus that colocalized with ΔFosB staining, suggesting increased vasopressin release in both groups. These results indicate that angiotensin signaling through the SFO contributes to increased water intake, but not dilutional hyponatremia, during congestive liver failure.

Region-Specific Changes in Transient Receptor Potential Vanilloid Channel Expression in the Vasopressin Magnocellular System in Hepatic Cirrhosis-Induced Hyponatraemia: Up-regulation of TRPV channels during hyponatraemia

The present study aimed to measure the expression of transient receptor potential (TRP) channels in the magnocellular neurones 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, which is a commonly used model of hepatic cirrhosis, associated with elevated plasma AVP. The present study tested the hypothesis that changes in TRP vanilloid (TRPV) channel expression may be related to inappropriate AVP release in BDL rats. To test our hypothesis, we utilised laser capture microdissection of AVP neurones in the PVN and SON and western blot analysis from brain punches. Laser capture microdissection and quantitative reverse transcriptase‐polymerase chain reaction demonstrated elevated TRPV2 mRNA in the PVN and SON of BDL 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 colocalisation counts of TRPV2 and oxytocin in both the magnocellular regions evaluated. In the SON but not the PVN, the transcription levels of TRPV4 were 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 rats. Our data suggest that regionally specific changes in TRPV expression in the magnocellular neurosecretory cell AVP neurones could alter their osmosensing ability.

Brain-Derived Neurotrophic Factor-Tyrosine Kinase B Pathway Mediates NMDA Receptor NR2B Subunit Phosphorylation in the Supraoptic Nuclei Following Progressive Dehydration: Dehydration and TrkB mediated NR2B phosphorylation

We studied the effects of water deprivation (WD) on the phosphorylation of tyrosine kinase B (TrkB) and NMDA receptor subunits in the supraoptic nucleus (SON) of the rat. Laser capture microdissection and quantitative reverse transcriptase polymerase chain reaction was used to demonstrate brain‐derived neurotrophic factor (BDNF) and TrkB gene expression in vasopressin SON neurones. Immunohistochemistry confirmed BDNF staining in vasopressin neurones, whereas staining for phosphorylated TrkB was increased following WD. 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 controls. 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 levels. To test whether increased phosphorylation of TrkB in the present 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. Although 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 WD. Thus, activation of TrkB in the SON following WD may affect cellular excitability through the phosphorylation of NR2B subunits.