Transcriptomic analysis of the basolateral amygdala (BLA) following post‐retrieval losartan on fear memory

Abstract

BackgroundA growing body of evidence has demonstrated the utility of angiotensin Receptor Blockers such as losartan in the treatment of maladaptive fear associated with posttraumatic stress disorder (PTSD). Our lab has previously shown the role of angiotensin type 1 receptor (AT1R) blockade in extinction fear memory, however its role in the alternative memory process of reconsolidation remain unclear. We hypothesized that post‐retrieval blockade of AT1R with losartan would disrupt reconsolidation and alter central transcriptomic pathways in the basolateral amygdala (BLA) ‐ a crucial site for emotional learning.MethodsBy combining classic Pavlovian auditory fear conditioning with whole‐genome RNA sequencing, we evaluated the effect of peripherally administered losartan (10mg/kg; i.p) on short‐term (STM) and long‐term (LTM) fear memory tests and at transcriptional level. Multiplex cytokine assays, RT‐qPCR and western blots were performed to analyze specific stress biomarkers and RAS components.ResultsMale C57Bl/6 mice injected with losartan after memory retrieval showed a significant reduction in freezing behavior in comparison to saline controls during LTM testing at 24hrs (F1, 17 = 5.739, p = 0.0284, n=12) and a trend for reduction during 1week (F1, 19 = 2.282, p =0.1474, n=12). No differences in freezing was observed a 2.5hr (STM) or in the absence of retrieval cue. An increased expression (2–3 fold; p<0.005) of immediate early genes; Fos, Arc and Egr‐1 was confirmed at 40mins in both the retrieval groups irrespective of the drug treatment. Using Illumina NextSeq series for RNA sequencing, at p<0.05, we identified 90 differentially expressed genes (DEGs) for saline and 38 for losartan relative to non‐retrieval (NR) group in BLA . At this early timepoint, a large overlap of 23 genes between the groups was observed and attributed to the behavioral intervention of fear memory consolidation. Independent of behavior, direct comparison between saline and losartan groups identified 13 unique DEGs at p<0.1 and one unique gene at p<0.05‐ low density lipoprotein receptor‐related protein 8 (Lrp8) involved in synaptic memory and plasticity. Among the uniquely expressed genes, only the losartan group comparison had differential expression for junction proteins Claudin‐5 and Gap junction beta‐6 protein with gene ontology results indicating an inhibition in biological adhesion (7.8% vs 2.3%) process. RT‐qPCR revealed no change in mRNA levels of RAS components by losartan treatment, with the exception of angiotensin converting enzyme (ACE) at 40mins post‐retrieval.ConclusionOur findings indicate that losartan, administered shortly after memory retrieval, reduces LTM but not STM, while altering the differential expression of specific genes in the BLA. These studies build upon growing evidence linking brain angiotensin receptors to fear‐related memory and suggest that targeting the AT1R may be a valid therapeutic approach during reconsolidation. Systemic AT1R inhibition therefore may be unique in its directional effects on fear memory, and important for the development of novel PTSD treatments.Support or Funding InformationNIH1R01HL137103‐01, AHA 15CSA24340001 , Illumina‐GWU Genomics Core Mini‐Grant.Differential BLA gene expression analysis following post‐retrieval losartan. (A)Volcano plots represents a gene with red dots having corrected p‐value < 0.05 and blue dots with pvalue >0.05 (B)Heat map representing genes which are differentially expressed after memory retrieval. Each line represents a DEG and blue and green indicate the low and high levels of expression respectivelyFigure 1