Transient receptor potential (TRP) proteins are non-selective cation channels that mediate sensory transduction. The neuroanatomical localization and the physiological roles of isoform TRPV2 in the rodent brain are largely unknown. We report here the neuroanatomical distribution of TRPV2 in the adult male rat brain focusing on hypothalamus and hindbrain regions involved in osmoregulation, autonomic function and energy metabolism. For this we utilized immunohistochemistry combined with brighfield microscopy. In the forebrain, the densest immunostaining was seen in both the supraoptic nucleus (SON) and the magnocellular division of the paraventricular nucleus (PVN) of the hypothalamus. TRPV2 immunoreactivity was also seen in the organum vasculosum of the lamina terminalis, the median preoptic nucleus and the subfornical organ, in addition to the arcuate nucleus of the hypothalamus (ARH), the medial forebrain bundle, the cingulate cortex and the globus pallidus to name a few. In the hindbrain, intense staining was seen in the nucleus of the solitary tract, hypoglossal nucleus, nucleus ambiguous, and the rostral division of the ventrolateral medulla (RVLM) and some mild staining in the area prostrema. To ascertain the specificity of the TRPV2 antibody used in this paper, we compared the TRPV2 immunoreactivity of wildtype (WT) and knockout (KO) mouse brain tissue. Double immunostaining with arginine vasopressin (AVP) using confocal microscopy showed a high degree of colocalization of TRPV2 in the magnocellular SON and PVN. Using laser capture microdissection (LCM) we also show that AVP neurons in the SON contain TRPV2 mRNA. TRPV2 was also co-localized with dopamine beta hydroxylase (DBH) in the NTS and the RVLM of the hindbrain. Based on our results, TRPV2 may play an important role in several CNS networks that regulate body fluid homeostasis, autonomic function, and metabolism.

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.

Chronic intermittent hypoxia (CIH) increases mean arterial pressure (MAP) and FosB/ΔFosB staining in central autonomic nuclei. To test the role of the brain RAS in CIH hypertension, rats were implanted with intracerebroventricular cannulae delivering losartan (ICVLOS: 1ug/hr) or vehicle (VEH) via mini‐osmotic pumps and telemetry devices for arterial pressure recording. A third group was given equimolar subcutaneous LOS (SCLOS) to differentiate between central and peripheral RAS effects. Rats were exposed to CIH for 7d then sacrificed for immunohistochemistry. ICV losartan had no effect on ΔMAP during CIH (VEH 5.9±0.8; ICVLOS 4.4±1.1; SCLOS 6.3±0.8 mmHg) but attenuated CIH‐induced increases in ΔMAP during the normoxic dark phase (VEH 6.7±0.8; ICVLOS 2.7±0.9; SCLOS 4.8±0.7 mmHg). Compared to VEH, ICVLOS and SCLOS attenuated FosB/ΔFosB staining in the organum vasculosum of the lamina terminalis (VEH 58±3; ICVLOS 23±6; SCLOS 37±7 cells/section). However, only ICVLOS attenuated staining in the median preoptic nucleus (VEH 31±5; ICVLOS 7±1; SCLOS 21±5 cells/section) and paraventricular (PVN) nucleus (VEH 32±7; ICVLOS 11±2; SCLOS 22± 3 cells/section). These data indicate that the central RAS contributes to the sustained, normoxic component of CIH‐induced hypertension and transcriptional activation of forebrain autonomic nuclei. Supported by P01 HL‐88052.

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