We tested whether ASIC1a disruption in mice reduced c-Fos expression in the basolateral amygdala (BLA) following exposure to single-shock context fear conditioning. We found that conditioning increased c-Fos expression in the BLA, and that ASIC1a disruption reduced the number of c-Fos positive cells post-conditioning (). In the amygdala, the ASIC1a effect was specific to the BLA, because c-Fos in the lateral, medial and central nuclei was normal in ASIC1a-null mice (Supplemental Fig. S1
). These results support the hypothesized effect of ASIC1a in the BLA, and bolstered our decision to target the BLA in subsequent experiments.
Figure 1 ASIC1a disruption attenuates c-Fos expression in the BLA. (A) Representative images of c-Fos immunohistochemistry at baseline and 1-h following fear conditioning in ASIC1a+/+ and ASIC1a–/– mice. (B) Analysis of c-Fos positive cell density (more ...)
To test the hypothesis that ASIC1a in the BLA is key for normal fear behavior, we produced an adeno-associated virus (AAV) encoding ASIC1a (AAV-ASIC1a) (See Methods) (Supplemental Fig. S2
). We targeted ASIC1a to the BLA in ASIC1a-null mice; mice expressing ASIC1a bilaterally in the BLA were defined as hits (AAVASIC1a-hit) (). Mice lacking ASIC1a expression in the BLA bilaterally were considered misses (AAV-ASIC1a-miss) () and were included in our behavioral analysis as a control for anatomic specificity. Mice injected with a similar vector expressing eGFP (AAV-eGFP) served as an additional negative control.
Two-weeks following injection, we used a 5-shock, context-fear-conditioning paradigm to examine fear behavior. As previously reported (Wemmie et al., 2003
), uninjected ASIC1a-null mice froze less than wild-type mice during training (). This deficit was not rescued in the AAV-ASIC1a-hit mice compared to the AAVASIC1a-miss, AAV-eGFP, and uninjected ASIC1a–/– controls.
The following day, we assessed the context-dependent fear response by returning the mice to the conditioning chamber for 5-min (minus footshocks). Strikingly, injection of AAV-ASIC1a in the BLA increased freezing relative to the other ASIC1a–/– groups and restored freezing to levels observed in wild-type mice (). Together these data suggest that ASIC1a expression in the BLA is sufficient to rescue the deficit in conditioned fear memory in ASIC1a-null mice, but not the normal freezing response to footshock during training.
The failure of BLA ASIC1a to rescue the ASIC1a–/– freezing deficit during training raised the question of whether it would rescue unconditioned fear (Rosen, 2004
). ASIC1a-null mice exhibit deficits in unconditioned fear responses to the predator odor trimethylthiazoline (TMT) (Coryell et al., 2007
. Unlike conditioned fear, such unconditioned fear behaviors are thought to depend more on the bed nucleus of the stria terminalis than the BLA (Walker et al., 2003
; Rosen, 2004
; Fendt et al., 2005
), although the BLA may play a role (Muller and Fendt, 2006
). Therefore, we hypothesized that expressing ASIC1a in the BLA would have little effect on TMT-evoked freezing in the ASIC1a-null mice. Indeed, the ASIC1a-null mice with ASIC1a targeted to the BLA did not differ from the other ASIC1a-null groups ().