PAC1 receptor modulation of freezing and flight behavior in periaqueductal gray

Abstract

The midbrain periaqueductal gray (PAG) region is a critical anatomical regulator of fear-related species-specific defensive reactions (SSDRs). Pituitary adenylate-cyclase-activating polypeptide (PACAP), and its main receptor PAC1, play an important role in fear-related behavior and anxiety disorders. However, the function of the PACAP-PAC1 system within the PAG with regards to SSDRs has received little attention. To address this gap, we used transgenic PAC1^flox/flox mice to examine both conditional and unconditional defensive reactions. We performed conditional PAC1 gene deletion within the ventrolateral(vl)PAG of PAC1^flox/flox mice using an adeno-associated virus (AAV) coding for Cre recombinase. Following viral expression, we used a white noise fear conditioning preparation that produces both an unconditional activity burst to the onset of noise that is followed by conditional freezing. On Day 1, mice received five white noise foot-shock pairings, whereas on Day 2, they were exposed to white noise five times without shock and we scored the activity burst and freezing to the white noise. Following behavioral testing, histology for immunofluorescent analysis was conducted in order to identify PACAP positive cells and stress-induced c-fos activity respectively. We found that PAC1 deletion in vlPAG increased the unconditional activity burst response but disrupted conditional freezing. PAC1 deletion was accompanied by higher c-fos activity following the behavioral experiments. Furthermore, a significant portion of PACAP-EGFP positive cells showed overlapping expression with VGAT, indicating their association with inhibitory neurons. The findings suggested that intact PACAP-PAC1 mechanisms are essential for SSDRs in vlPAG. Therefore, midbrain PACAP contributes to the underlying molecular mechanisms regulating fear responses.

Keywords: PAC1 receptor; VGAT; darting; early gene expression; fear conditioning; freezing; periaqueductal gray.

FIGURE 1

Baseline freezing activity for Day 1 and Day 2. There is no significant difference between groups for both day 1 and day 2 baseline freezing activity. n = 6–8 per group.

FIGURE 2

White noise‐evoked freezing activity. Analysis obtained from two‐way ANOVA showed there is a significant trial effect **p = 0.0043, but no significant group difference on Day 1. In figure b, two‐way ANOVA showed a significant trial effect *p < 0.05 and significant group difference **p = 0.0052, mean data showed a significant group difference for the freezing on Day 2; *p < 0.05. n = 6–8 per group.

FIGURE 3

White noise‐evoked activity burst. Activity burst based on the formula (preWN/(preWN + WN)) is calculated for day 1 and day 2. Results showed there is a significant trial effect *p < 0.05 on Day 1, whereas analysis obtained from Day 2 results showed a significant group difference **p = 0.0033, mean data showed significant difference between groups *p < 0.05. n = 6–8 per group.

FIGURE 4

Velocity measure (activity bursts per minute). Analysis obtained from ANOVA showed there is a significant group difference *p < 0.05, mean data showed a marginal significant difference p = 0.0504 between groups on Day 2. n = 5–7 per group.

FIGURE 5

The relationship between freezing and peak activity ratio. Analysis obtained from Pearson correlation showed there is no significant relationship between the freezing response and the activity burst p > 0.05 for the PAC1^flox/flox Cre group, whereas a significant relationship was observed p = 0.003 for PAC1^flox/flox GFP group.

FIGURE 6

Ventrolateral PAG c‐fos expression. Analysis obtained from ANOVA showed there is a significant group difference *p < 0.05. Midline figure shows bilateral AAV expression (green) in the vlPAG. Figure at the right side shows a high magnitude image of the AAV expression (green) and c‐fos expression (red) at the vlPAG. n = 6–8 per group.

FIGURE 7

PACAP‐EGFP positive cells overlapping with VGAT positive cells in ventrolateral PAG. (A) The exemplifier data shows VGAT cells (red) overlapping with EGFP cells (green) under the condition with the primary and secondary antibodies used together for the double staining. However, the middle histogram data shows no VGAT positive cells due to the absence of primary antibody for VGAT. Rather, the primary antibody for EGFP is used and the secondary antibody is used for both EGFP and VGAT. (B) One‐way ANOVA analysis showed there is no difference between VGAT cells, EGFP cells and VGAT + EGFP cells. n = 5 per group. (C) Number of cells overlapping for VGAT and PACAP. n = 5.