Figure 8.

Recovery of Fear Conditioning Memory after NeuroD1 Treatment

(A) Experimental design of fear conditioning test in rats. ET-1 or saline was injected into the BLA, followed by fear conditioning 3 weeks later. Fear memory tests were performed before viral injection and 3 weeks after viral injection to assess the retention of fear memory. Right two panels illustrate the amygdala lesion induced by the infusion of ET-1. Gray areas represent the minimum (dark) and maximum (light) spread of the lesion across different anterior-posterior levels of BLA (−2.12, −2.56, and −2.80 from bregma), condensed in one level for illustration. CeA, central nucleus of the amygdala, op., optical tract. (B) ET-1 lesion reduced freezing during fear conditioning (F_(2,31) = 3.98, *p = 0.02, day 21) at both ET-1/Control (blue, *p = 0.021, n = 10) and ET-1/NeuroD1 (magenta, *p = 0.019, n = 14) groups, compared to saline/saline group (black, n = 10). Reduced freezing (F_(2,31) = 3.45, *p = 0.044) was also observed on the next day (day 22) in both ET-1/Control (p = 0.030) and ET-1/NeuroD1 (*p = 0.031) groups. Rats were then infused with control virus or NeuroD1 and re-tested 3 weeks later (F_(2,31) = 5.86, *p = 0.006, day 45). In the ET-1/NeuroD1 group, freezing returned to the levels of the Saline/Saline group (*p = 0.81), and was significantly higher than ET-1/Control group (*p = 0.004). “x” denotes baseline pre-tone freezing levels. Hab, habituation; Cond, conditioning; pre-CS, pre-conditioned stimulus. One-way ANOVA followed by Duncan’s post hoc test. Data are expressed as mean ± SEM in blocks of two trials. *p < 0.05. (C) After fear conditioning test, immunostaining of rat brain sections confirmed the injection of NeuroD1-GFP viruses into the BLA (green, left panel), and the NeuroD1-infected cells were mostly NeuN-positive neurons (right panels). Scale bar, 1,000 μm.