Aplnr knockout mice display sex-specific changes in conditioned fear

Abstract

The G-protein-coupled receptor APLNR and its ligands apelin and ELABELA/TODDLER/apela comprise the apelinergic system, a signaling pathway that is critical during development and physiological homeostasis. Targeted regulation of the receptor has been proposed to treat several important diseases including heart failure, pulmonary arterial hypertension and metabolic syndrome. The apelinergic system is widely expressed within the central nervous system (CNS). However, the role of this system in the CNS has not been completely elucidated. Utilizing an Aplnr knockout mouse model, we report here results from tests of sensory ability, locomotion, reward preference, social preference, learning and memory, and anxiety. We find that knockout of Aplnr leads to significant effects on acoustic startle response and sex-specific effects on conditioned fear responses without significant changes in baseline anxiety. In particular, male Aplnr knockout mice display enhanced context- and cue-dependent fear responses. Our results complement previous reports that exogenous Apelin administration reduced conditioned fear and freezing responses in rodent models, and future studies will explore the therapeutic benefit of APLNR-targeted drugs in rodent models of PTSD.

Keywords: Apelin; PTSD; behavior; conditioned fear; receptor.

Fig. 1.. Behavioral testing timeline.

Mice (10–12 weeks in age at the beginning of the study) underwent a battery of behavioral tests, with order planned so that more stressful procedures (conditioned fear) and single housing (2-bottle choice test for sucrose preference) occurred at the end of the study. NORT = novel object recognition test; ASR = acoustic startle response; PPI = pre-pulse inhibition.

Fig. 2.. No genotype differences in body weight, activity, or motor coordination between Aplnr wild-type and knockout mice across the behavior study.

Data are means ± SEM) for each group. A, B) Age is approximate number of weeks. C, D) Locomotor activity and rearing movements during a one-hour test in a novel open field. E) Latency to fall from an accelerating rotarod. Maximum trial length was 300 sec. Trials 4 and 5 were given 48 hours after the first three trials.

Fig. 3.. Magnitude of acoustic startle responses and prepulse inhibition.

Data shown are means (+ SEM) for each group. Trials included no stimulus (No S) trials and acoustic startle stimulus (AS; 120 dB) alone trials. *p < 0.05.

Fig. 4.. Increased freezing responses in male Aplnr KO mice during tests for contextual learning.

Data are means (+ SEM) for a 5-min test. Test 1 was conducted 24 hr after training. The second test for contextual learning (Test 2) was conducted 2 weeks following Test 1. *p < 0.05; **p < 0.01.

Fig. 5.. Increased freezing responses in male Aplnr KO mice during tests for cue-dependent learning.

Data are means (+ SEM) for a 5-min test. Test 1 was conducted 48 hr after training. The second test for cue learning (Test 2) was conducted 2 weeks following Test 1. During each test, a 3-min, 80 decibel acoustic stimulus was presented 2 min after mice were placed in modified conditioned fear chambers. *p < 0.05; **p < 0.01.

Fig. 6.. Differential expression of Aplnr in the dentate gyrus between male and female mice.

Aplnr-driven Cre recombinase mice were crossed to a tdTomato cre reporter strain (Jackson Labs Strain 7909). A–B: dentate gyrus of male mice. C–D: dentate gyrus of female mice. Tissues were frozen in OCT, and 10 μm sections were taken and stained with DAPI. Fluorescent images were captured at 10X magnification using a Texas Red filter on an Olympus DP70 camera system mounted to an Olympus IX51 fluorescence microscope. Images were captured with identical settings and conditions across mice.