Acute stressor exposure increases anxiety-like behavior and c-Fos expression in LC neurons projecting to CeA. The experimental timeline is shown in A. Animals underwent a surgical procedure to inject two distinct retrograde tracers in mPFC (red; shown in magenta) and CeA (green). Seven days later, they underwent stressor exposure (n = 7) or control conditions (n = 7). Behavior was then assessed in the EPM, and 2 h later rats were sacrificed for immunofluorescent detection of c-Fos in LC. Time spent in the open arms was square-root transformed to satisfy requirements for parametric statistical testing and was significantly decreased in stressed rats (gray bars with filled circles) relative to control rats (white bars with open circles; B; shown as median with interquartile range). Stressed rats also spent more time freezing than control rats (C). Mean heat maps for behavior in the EPM are shown in D. Injections of retrograde tracers resulted in similar numbers of labeled cells projecting to mPFC and CeA regardless of treatment. The number of double labeled cells projecting to both regions (dual) was small (E). The number of c-Fos positive nuclei was logarithm transformed to satisfy requirements for parametric testing, and was found to significantly increase in both the LC→CeA and LC→mPFC projections in response to stressor exposure. However, the number of c-Fos positive nuclei was significantly greater in the LC→CeA projection than LC→mPFC projection in stressed animals (F; shown as median with interquartile range). A representative photomicrograph of labeled neurons in the dorsal third of LC is shown in G. LC→mPFC neurons are magenta, LC→CeA neurons are green, and c-Fos is shown in blue. White arrowhead and arrow indicate examples of c-Fos+ and c-Fos- LC→CeA neurons, respectively. Yellow arrowhead and arrow indicate examples of c-Fos+ and c-Fos- LC→mPFC neurons, respectively. Scale bar = 25 μm. Representative images of injection of tracers into CeA and mPFC are shown H and I, respectively. *: p < 0.05. †: p < 0.05 after square root transformation. ‡: p < 0.05 after logarithm transformation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)