NMDA Receptor Plasticity in the Hypothalamic Paraventricular Nucleus Contributes to the Elevated Blood Pressure Produced by Angiotensin II

Abstract

Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulation of the hypothalamic paraventricular nucleus (PVN). Many forms of glutamate-dependent plasticity are mediated by NMDA receptor GluN1 subunit expression and the distribution of functional receptor to the plasma membrane of dendrites. Here, we use a combined ultrastructural and functional analysis to examine the relationship between PVN NMDA receptors and the blood pressure increase induced by chronic infusion of a low dose of Ang II. We report that the increase in blood pressure produced by a 2 week administration of a subpressor dose of Ang II results in an elevation in plasma membrane GluN1 in dendrites of PVN neurons in adult male mice. The functional implications of these observations are further demonstrated by the finding that GluN1 deletion in PVN neurons attenuated the Ang II-induced increases in blood pressure. These results indicate that NMDA receptor plasticity in PVN neurons significantly contributes to the elevated blood pressure mediated by Ang II.

Keywords: gene deletion; gene therapy; hypertension; hypothalamus; synaptic plasticity.

Figure 1.

Mice with Ang II-induced elevations in blood pressure show an increase in plasma membrane GluN1 in dendritic profiles also labeled for nNOS. Electron micrographs from the PVN of mice that received vehicle (A) or Ang II (B) showing silver-intensified immunogold labeling for GluN1 and immunoperoxidase reaction product for nNOS (white arrows) in dendritic profiles. Immunogold labeling for GluN1 is located in the cytoplasm (arrow) and near the surface (circles). Immunoperoxidase labeling for nNOS is diffuse throughout the cytoplasm or localized to endomembranes. There was a significantly higher density of total (C) and surface (D), but not cytoplasmic (E), GluN1 labeling in dendritic profiles of PVN neurons in Ang II-treated animals compared with those administered saline. GluN1 + nNOS, dual-labeled dendrite; Un-Te, unlabeled terminal; curved arrow, asymmetric synapse. Data are presented as mean ± SEM; ***p < 0.0001. Scale bars, 500 nm.

Figure 2.

The Ang II-induced increase in blood pressure is associated with altered plasma membrane levels of GluN1 in single-labeled dendrites. Electron micrographs showing silver-intensified immunogold labeling for GluN1 in dendritic profiles from the PVN of mice that received vehicle (A) or Ang II (B). GluN1 labeling is prominently found in the cytoplasmic compartment (arrows) and the plasma membrane (circles). There was a significantly higher density (particles per unit area) of total (C) and surface (D), but not cytoplasmic (E), GluN1 labeling in mice administered Ang II compared with vehicle controls. nNOS-d, nNOS-labeled dendrite; GluN1-d, GluN1-labeled dendrite. Data are presented as mean ± SEM; *p < 0.01. Scale bars, 500 nm.

Figure 3.

Microinjection of rAAV-Cre into the PVN of fGluN1 mice results in a decrease in GluN1 gene expression. Unilateral microinjection of rAAV-Cre in the PVN resulted in expression of the reporter protein GFP (A) and reduced GluN1 mRNA levels (B) in the injected area. Expression of the neuronal marker NeuN is shown in an adjacent section (C). There was a significant reduction in GluN1 (D) but not NeuN (E) labeling calculated as the ratio of the number of labeled cells ipsilateral and contralateral to the microinjection site; *p < 0.05. Scale bars: A, 1 mm; B, C, 0.5 mm.

Figure 4.

Deletion of GluN1 in PVN neurons suppresses NMDA receptor-mediated NO and ROS production. Representative micrograph of dissociated PVN neurons (A) obtained from a mouse that received unilateral rAAV-Cre in the PVN, demonstrating a reduction in GluN1 labeling (B). The production of NO as measured by DAF-FM in these cells did not significantly differ between vehicle (Veh; 0 μm NMDA) and 100 μm NMDA (C–E), demonstrating that the GluN1 deletion attenuated NMDA-dependent NO production. Control cells (Non-KD) showed the expected increase in NMDA-mediated DAF-FM intensity (E). There was also a reduction in NMDA-mediated ROS production as measured by DHE staining; *p < 0.05. Scale bar, F, 20 μm.

Figure 5.

PVN GluN1 deletion results in a significant attenuation of Ang II-induced elevations in blood pressure. A, Daily radiotelemetry measurements of MAP during infusion of Ang II in fGluN1 mice microinjected with PVN-targeted AAV encoding Cre-recombinase (rAAV-Cre) or control rAAV-GFP. N = 6–7. B, The maximum MAP occurred during days 7–14 of Ang II treatment. C, An example of labeling for the GFP reporter in a mouse that received bilateral rAAV-Cre. The PVN is indicated by the areas bounded by the curved dashed figures; *p < 0.05. Scale bar, 1 mm.