Evaluation of a brain angiotensinergic circuit in fear‐related behavior

Abstract

BackgroundThe renin‐angiotensin system (RAS) has been implicated in post‐traumatic stress disorder (PTSD), however the mechanisms responsible for this connection and the therapeutic potential of targeting the RAS in PTSD remains unknown. Combining neuroanatomical, genetic knockdown and behavioral approaches, we examined components of the brain RAS, in particular the origin of brain angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R) and their functional role in fear‐related behavior.MethodsRNA Scope combined with immunohistochemistry (IHC) were used to examine the expression pattern and cellular localization of brain AGT in C57BL/6J mice. Dual IHC was used to characterize AT1R‐eGFP+ cells in the amygdala of the AT1R‐eGFP‐BAC reporter mouse. Pavlovian fear conditioning and cre‐expressing lentivirus (LV) injection were used to demonstrate the effects of AT1R deletion on fear memory in male AT1R‐floxed mice.ResultsAGT mRNA was primarily expressed in the diencephalon, midbrain and hindbrain, including the parabrachial complex (PB) which sends strong neuropeptide projections to the amygdala. Some AGT positive cells in PB were determined to be of neuronal origin as they co‐localized with neuronal marker NeuN (FIG 1A). In the amygdala, AGT mRNA was highly expressed in the medial division of the central amygdala (CeM) and the medial amygdala (MeA), but not in the lateral division of the central amygdala (CeL) or basolateral amygdala (BLA) (FIG 1B). Different from AGT, the AT1R‐eGFP+ neurons in the amygdala were predominantly expressed in CeL, with little AT1R‐eGFP expression in BLA, MeA or CeM (FIG 1C). Characterization of AT1R‐eGFP+ neurons in the CeL demonstrated that the AT1R neurons are GABAergic (98.6%) and the majority were co‐located with fear off neuronal marker protein kinase C‐δ (65.7%) as opposed to the fear on neuronal marker somatostatin (9.4%). Next, using cre‐expressing lentivirus, AT1R was deleted from the central amygdala (CeA) of AT1R‐floxed mice. CeA AT1R deletion did not affect fear acquisition, but enhanced the extinction of fear as determined by a reduction in percent freezing during extinction training (57.04% ± 3.9 LV‐GFP v.s. 42.6% ± 4.1 LV‐Cre group, p<0.05, n=9) and retention tests (53.5% ± 5.3 LV‐GFP v.s. 34.4% ± 5.3 LV‐Cre group, p<0.05, n=9) (FIG 2).ConclusionsThese findings provide neuroanatomical and behavioral evidence for brain RAS pre‐cursor AGT and the AT1R in the extinction of fear memory. We speculate that AGT modulates the PB‐CeA circuit during threat responding in fear learning and memory. To further understand the neuroanatomy and function of this circuit, future studies are needed to apply retrograde tracing approaches combined with AGT RNA Scope and region specific deletion of the AGT positive neurons.Distribution of AGT and AT1R in PB and Amygdala Subdivisions(A) AGT mRNA was found in PB (left) and co‐localized with NeuN (right). (B) AGT mRNA was mainly expressed in CeM and MeA. (C) AT1R‐GFP+ neurons were predominately found in CeL.Figure 1Enhanced fear extinction following CeA AT1R deletion.(A) Auditory fear conditioning protocol. (B) AT1R deletion from the CeA did not affect fear acquisition (left) but enhanced the extinction of fear as shown by reduced percent freezing during extinction training (middle) and extinction retention test (right).Figure 2