Fear Conditioning & Circadian Activity

Figure 1: Fear Conditioning Alters Circadian Activity Patterns

• Fear conditioning affects circadian locomotor activity differently based on the conditions

Light Fear (LF) vs. Dark Fear (DF) Comparison

• Actograms show that LF mice have minor activity shifts but remain mostly nocturnal
• There is a slight increase in daytime activity for LF mice
• DF mice show a stronger shift with pronounced activity during the day, even in constant darkness
• Activity profiles indicate LF mice maintain nocturnal preference but increase daytime activity post-fear conditioning
• DF mice show a more robust shift, with fear training having a greater impact on circadian adaptation
• Fear conditioning significantly increases activity during the day, especially in DF mice
• Fear experienced at night results in stronger entrainment to circadian rhythms in mice
• Rayleigh plots reveal greater shifts in activity onset in DF mice (~3-4 hours) compared to LF mice (~1-2 hours)
• This confirms fear conditioning is more effective during the dark phase
• Dark-phase fear training (DF) leads to stronger circadian disruptions than light-phase fear (LF)
• DF mice maintain altered activity patterns even in constant darkness

Cued Fear vs. Non-Cued Fear Comparison

• Cued fear results in structured locomotor activity rhythms
• Activity clearly adapts to the fear-associated time window in cued fear conditioning
• Non-cued fear causes fragmented and erratic activity patterns
• Entrainment effect is weaker in non-cued fear conditioning
• Cued fear leads to a predictable activity shift toward the safe phase
• Non-cued fear results in scattered and inconsistent behavioral adaptations after fear training
• Cued fear significantly increases activity during the safe period
• Reinforces predictable fear stimuli enhance circadian entrainment more than random shocks
• Cued fear induces a stronger and more consistent phase shift compared to non-cued fear
• Non-cued fear produces highly variable and weak shifts in activity onset
• Cued fear (predictable stimuli) effectively entrains circadian rhythms
• Non-cued fear disrupts circadian rhythms leading to erratic behavioral adaptation

Figure 2: Circadian Gene Expression in the SCN Following Fear Conditioning

Measures whether fear conditioning alters core circadian gene expression in the SCN

• Fear conditioning doesn't significantly alter mPer1 expression in the SCN

• Rhythmic oscillations remain stable across both LF and DF groups

• Spatial expression patterns stay intact, confirming mPer1 expression is not disrupted by fear

• mBmal1 exhibits normal oscillations, with peaks and troughs occurring at expected circadian time points

• No phase shift is observed due to fear conditioning

• No spatial alterations are found, indicating transcriptional regulation of Bmal1 remains unaffected in fear conditioning

• Fear conditioning does not significantly disrupt the molecular clock in the SCN

• Reinforces that behavioral adaptations occur through circadian entrainment rather than core clock gene regulation

Figure 3: Role of SCN-Specific Bmal1 in Fear Entrainment

• The main point here is to find out whether Bmal1 is necessary for fear-induced circadian adaptations

Findings Across Bmal1 Genotypes

• Cami-Bmal1+/+ (Wild-type) mice show robust circadian rhythms, with clear post-fear conditioning shifts toward the safe phase
• Cami-Bmal1+/- (Heterozygous) mice exhibit intermediate entrainment, showing some phase shift but reduced rhythmic amplitude
• Cami-Bmal1-/- (Knockout) mice display disrupted and fragmented activity rhythms, failing to entrain to the fear stimulus

Panel Breakdown

• Actograms in WT and heterozygous mice show strong post-fear entrainment, while knockouts fail to modify their activity patterns.
• Fear conditioning increases daytime activity in WT and heterozygous mice, but not in knockouts
• WT and heterozygous mice shift activity to the safe phase, while knockouts exhibit no behavioral adaptation
• WT and heterozygous mice strengthen rhythmicity post-conditioning, but knockouts fail to show circadian reinforcement
• Bmal1 is necessary for fear-driven circadian entrainment
• Knockout mice fail to adapt behaviorally, confirming Bmal1 is requires for entrainment in excitatory neurons

Final Conclusion

• Bmal1+/+: Fear-entrained rhythms are intact, with robust behavioral adaptation
• Bmal1+/-: Partially maintains rhythmic adaptation but with weaker responses than wild-type
• Bmal1-/-: Fails to entrain to fear stimuli, confirming that Bmal1 is essential for integrating fear with circadian behavior

Figure 4: Role of SCN and Extra-SCN Bmal1 in Fear Entrainment

• Identifies whether the SCN alone is sufficient for fear-driven circadian adaptation

Key Findings

• The SCN targeted Bmal1 deletion weakens rhythmicity but does not fully eliminate entrainment
• This indicates that peripheral oscillators contribute to fear-driven circadian behavior
• Locomotor activity in knockouts display weaker but still detectable rhythmicity
• The SCN is important but not solely responsible for fear-driven circadian adaptation
• Fear enhances circadian amplitude in wild-type, SCN-Bmal1+/+ mice but not in SCN knockouts
• This confirms SCN’s role in rhythmic reinforcement after fear conditioning
• The SCN-intact mice retain strong fear-driven rhythms
• This suggests that peripheral oscillators are not required for fear entrainment
• Fear-driven rhythms persist when Bmal1 is deleted outside the SCN, reinforcing SCN sufficiency
• Phase shifts occur only in SCN-intact mice, further proving that the SCN is the primary driver of fear entrainment

Conclusion

• SCN Bmal1 is necessary for amplifying fear-driven rhythms, but peripheral oscillators play only a minor role
• The SCN alone is sufficient to sustain fear entrainment

Final Takeaways & Comparative Summary

• DF causes stronger circadian disruptions than LF, persisting even in constant darkness
• Cued fear effectively entrains circadian behavior, whereas non-cued fear disrupts it
• Fear conditioning does not alter SCN core clock genes (mPer1, Bmal1)
• This suggests behavioral adaptations occur via entrainment rather than molecular changes in the brain
• WT and heterozygous mice successfully adapt to fear-induced circadian shifts, but Bmal1 knockouts fail to entrain SCN-specific Bmal1 knockouts show weaker rhythmicity, but retain some fear-driven adaptation
• This indicates peripheral oscillators play a secondary role in fear conditioning
• Extra-SCN Bmal1 deletion has no major effect on fear entrainment
• Proves the SCN is sufficient for sustaining fear-driven circadian rhythms
• Fear modulates circadian behavior through SCN-driven mechanisms
• Bmal1 is required for fear-induced entrainment
• Dark-phase fear and cued conditioning producing the strongest effects
• The SCN alone is capable of sustaining fear-driven circadian adaptation