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

• Published in: Journal of neuroendocrinology 2011-10, Vol.23 (10), p.894-905
• Main Authors: Carreño, F. R., Walch, J. D., Dutta, M., Nedungadi, T. P., Cunningham, J. T.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; Blotting, Western; Brain-derived neurotrophic factor; Brain-Derived Neurotrophic Factor - metabolism; Dehydration; Dehydration - metabolism; DNA Primers; Excitability; Fundamental and applied biological sciences. Psychology; Fusion protein; Glutamic acid receptors; Glutamic acid receptors (ionotropic); Immunohistochemistry; Lasers; N-Methyl-D-aspartic acid receptors; NMDA receptor; osmotic pressure; Phosphorylation; Polymerase chain reaction; Protein-tyrosine kinase; Protein-Tyrosine Kinases - metabolism; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate - metabolism; Rehydration; Reverse Transcriptase Polymerase Chain Reaction; RNA-directed DNA polymerase; Serine; Supraoptic nucleus; Supraoptic Nucleus - enzymology; Supraoptic Nucleus - metabolism; TrkB receptors; Vasopressin; Vertebrates: endocrinology; Western blotting
• ISSN: 0953-8194; 1365-2826
• DOI: 10.1111/j.1365-2826.2011.02209.x

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.