Reporter mouse strain provides a novel look at angiotensin type-2 receptor distribution in the central nervous system

Abstract

Angiotensin-II acts at its type-1 receptor (AT1R) in the brain to regulate body fluid homeostasis, sympathetic outflow and blood pressure. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of limited ability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual immunohistochemistry (IHC)/ ISH studies conducted in AT2R-eGFP reporter mice found that eGFP and AT2R mRNA were highly co-localized within the brain. Qualitative analysis of eGFP immunoreactivity in the brain then revealed localization to neurons within nuclei that regulate blood pressure, metabolism and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]), as well as limbic and cortical areas known to impact stress responding and mood. Subsequently, dual IHC/ISH studies uncovered the phenotype of specific populations of AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-1 (80.3 ± 2.8 %), while a smaller subset express vesicular glutamate transporter-2 (18.2 ± 2.9 %) or AT1R (8.7 ± 1.0 %). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular nucleus of the hypothalamus (PVN), eGFP- immunoreactivity is localized to efferents terminating in the PVN and within GABAergic neurons surrounding this nucleus. These studies demonstrate that central AT2R are positioned to regulate blood pressure, metabolism and stress responses.

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

“…They reported very limited overlap between the brain expression of AT 1A and AT 2 receptor mRNAs. de Kloet et al (2014) used bacterial artificial chromosome transgenic AT 2 receptor-enhanced green fluorescent protein (eGFP) reporter mouse to localize AT 2 receptors at a cellular level. The authors examined for colocalized eGFP and AT 2 receptor mRNA within the State of the Angiotensin Receptors brain.…”

Angiotensin Type-2 Receptors Influence the Activity of Vasopressin Neurons in the Paraventricular Nucleus of the Hypothalamus in Male Mice

“…For additional details, see the vendor's website: www.mmrrc.org. This mouse line was previously characterized (12), and it was determined that the distribution of AT2R-eGFP in the mouse brain is comparable to the AT2R binding observed using autoradiography (18). Mice used for the studies were backcrossed onto a C57BL/6J background for 5ϩ generations and were hemizygous for the transgene.…”

“…Using recent advances in molecular and genetic techniques to localize AT2R within the brain with a high degree of sensitivity and resolution, we have previously determined that AT2R positive cell bodies and nerve terminals are well-positioned to influence brain regions that regulate neuroendocrine axes (12). One critical site of Ang-II actions within the brain is the paraventricular nucleus of the hypothalamus (PVN), which is an integrative region for the control of several homeostatic systems and densely expresses the AT1R (13).…”

“…Of relevance, while the PVN itself does not contain any AT2R-positive neuronal cell bodies, nerve fibers and/or terminals arising from AT2R-positive cells are densely-localized to the parvocellular and magnocellular portions of the PVN. Further, we have previously determined that these AT2R-positive fibers/terminals come into close proximity to AVP neurons within this area and that many of these AT2R-positive neurons originate from brain regions that send inhibitory projections to the PVN, including the preoptic area and GABAergic neurons that lie adjacent to the PVN (12).…”

Control of Energy Balance by the Brain Renin-Angiotensin System

“…In addition, mice with global AT2 receptor gene disruption exhibit reduced body weight and adipose gain during high-fat feeding in part through reduced food intake [69]. Exciting new visualization methods from de Kloet et al demonstrate localization of AT2 gene expression specifically in GABAergic and cholinergic neurons within brain regions that are known to be involved with the control of ingestive behaviors (appetite/taste/taste preference/malaise) such as the NTS, median preoptic nucleus (MnPO), and area postrema (AP) [70].…”