Experiment B CP during fear acquisition normalizes
Experiment 4B: CP154,526 during fear acquisition normalizes fear-potentiated startle and prevents exacerbation of contextual conditioned fear in SERT−/− rats.
As genotypes differed markedly in basal fear acquisition, drug effects could differ strongly between genotypes. Therefore, all effects in this experiment were analyzed separately for each genotype. In SERT+/+ and SERT+/−, administration of CP154,526 during acquisition had no effect on fear IL-18, human recombinant protein 24h later, nor on the startle response per se (SERT+/+: [drug×trial interaction F2,32<1, overall effect of drug F2,32<1]; SERT+/−: [drug×trial interaction F2,37<1, overall effect of dose F2,37=1.1, NS]; Figure 4D and E, respectively). In SERT−/−, on the other hand, CP154,526 treatment during acquisition had a differential effect on cued and non-cued trials ([drug×trial interaction F2,33=3.8, p<0.05]; Figure 4F). Administration of 10mg/kg CP154,526 during acquisition resulted in a significant potentiation of the startle response in response to cued trials (effect trial: [T11, p<0.01]), whereas the response to cued and non-cued trials was similar following treatment with vehicle or 30mg/kg CP154,526 (effect trial: vehicle [T10=0.3, NS]; 30mg/kg [T12=−0.2, NS]). This significant fear potentiation following 10mg/kg CP154,526 in SERT−/− was reflected in the absolute difference scores, as these scores were significantly increased compared to SERT−/− vehicle controls (Effect of drug [F2,33=3.8, p<0.05]; post-hoc analyses: 10mg/kg vs vehicle [p=0.033], 30mg/kg vs vehicle [p=0.913], inset Figure 4F), whereas they were unaltered in SERT+/+ and SERT+/− rats [effect of drug F2,32<1 and F2,37<1 respectively]. In addition, in SERT−/−, a main effect of CP154,526 on overall startle responding revealed that CP154,526 administration during acquisition also prevented the development of contextual conditioned fear, as measured 24h later (main effect drug [F2,33=4.6, p<0.05]). This effect was primarily due to the 30mg/kg dose.
Experiment 5: CP154,526 does not affect expression of fear-potentiated startle.
CP154,526 had no specific effect on the level of fear potentiation when administered before the fear-potentiated startle test in pre-trained rats, rather it significantly decreased the overall startle response (main effect drug [F1,47=21.5, p<0.001], dose×condition×trial interaction [F1,47<1]); Figure 5). This effect tended to be stronger in the cue-shock group, compared to the cue-no-shock group (drug×condition interaction [F1,47=3.86, p=0.055]). The genotype differences in expression of FPS, as detected earlier, were confirmed (trial×genotype interaction in cue-shock group [F2,25=10.0, p=0.001]; effect trial in SERT+/+ [F1,9=18.2, p<0.01], SERT+/− [F1,8=19.0, p<0.01] and SERT−/− F1,8=1.4, NS]). In the cue-no-shock control group, on the other hand, no fear potentiation was present and genotypes did not differ in startle reactivity (effect of trial [F1,22<1], effect of genotype [F2,22<1], genotype×trial interaction [F2,22=2.7, p<0.1]).
Experiment 6: CRF1 receptor mRNA expression in BLA and DR was not significantly altered in SERT−/− rats.
CRF1 receptor levels in the basolateral amygdala, represented by number of dots, were not significantly different between SERT+/+ and SERT−/− rats (main effect genotype [F1,16=1.3, NS], Figure 6A). Because of a trend towards a difference between hemispheres (main effect hemisphere [F1,16=4.1, p<0.1], genotype×hemisphere interaction [F1,16=3.4, p<0.1]), genotype effects were also analyzed in both hemispheres separately. This analysis showed a significant decrease in CRF1 receptor levels in the left BLA of SERT−/− rats (t16=−2.468, p<0.05), whereas CRF1 receptor levels in the right BLA did not differ between genotypes (t16=−0,116, NS). No significant differences CRF1 receptor levels in the dorsal raphe nucleus were found between SERT+/+ and SERT−/− rats ([t18=0.125, NS], Figure 6B).