High Salt Intake Increases Contextual Fear Retrieval and Synaptic Inhibition of Medial Entorhinal Cortex‐Projecting Neurons of the Basolateral Amygdala

Abstract

Fear-related neuropsychiatric disorders are becoming increasingly prevalent. Although the impact of psychological stress on fear disorders is well studied, less attention has been paid to the impact of physiological stressors. High salt (NaCl) intake is a common physiological stressor, well known to increase the prevalence and severity of cardiovascular disease (CVD), but effects on fear-related mental health disorders, which are often co-morbid with CVD, have only recently been investigated. Our lab reported that mice consuming excess salt exhibit anxiety-like active coping behaviors and heightened basolateral amygdala (BLA) neuronal activation following swim stress. BLA principal neurons (PN) are critical in processing fear memory and their projections to medial entorhinal cortex (BLA-mEC) are implicated specifically in contextual fear memory. Thus, we formulated and tested the hypothesis that high salt intake increases contextual fear memory retrieval linked to hyper-activation of BLA PNs following retrieval testing. Because BLA PNs are normally under strong control by local interneurons (INs), we further hypothesized that high salt intake reduces ongoing synaptic inhibition of BLA-mEC PNs. To increase salt intake, normal drinking water was replaced with a solution of 4% NaCl for 7 consecutive days. Immediately thereafter, contextual fear conditioning was performed and 24 h later mice were re-exposed to the same context for memory retrieval with fear quantified as percentage of time spent posturally immobile (i.e., freezing behavior). Ninety min after fear retrieval testing, mice underwent perfusion fixation and Fos immunostaining of BLA. In a separate treatment group, mice received rhodamine retrobead injections in the mEC to identify BLA-mEC neurons in brain slices prepared 3-5 days later. Results indicate that high salt intake (HSI) increased fear memory retrieval compared to normal salt intake (NSI) (HSI: 69 ± 5% (n=9), NSI: 44 ± 6% (n=9), unpaired t-test P=0.0038) without changing the mean intensity of Fos immunofluorescence in BLA (HSI: 220 ± 20 F/F0 (n=6), NSI: 240 ± 15 F/FBKGD (n=6), unpaired t-test P=0.4412). Compared to NSI, HSI increased the amplitude (HSI: 32 ± 0.6 pA (n=7), NSI: 22 ± 0.4 pA (n=9), unpaired t-test P<0.0001) and frequency (HSI: 5.3 ± 1.4 Hz (n=7), NSI: 2.5 ± 0.8 Hz (n=9), unpaired t-test P=0.0415) of spontaneous inhibitory postsynaptic currents recorded from BLA-mEC neurons. Thus, HSI enhances retrieval of contextual fear memory despite an apparent increase of inhibitory synaptic control of BLA-mEC PNs and lack of evidence of greater overall BLA neuronal transcriptional activation. Whether greater synaptic inhibition is maintained in vivo to buffer HSI-induced excitatory drive remains to be determined. Presently, our findings are consistent either with HSI acting downstream of BLA to enhance contextual fear memory retrieval or that Fos immunoreactivity does not accurately index strengthening of excitatory input to BLA caused by HSI. Whichever the case might be, findings warrant further investigation into the underlying mechanisms of HSI-induced enhancement of fear memory retrieval.