Limits...
A fear-inducing odor alters PER2 and c-Fos expression in brain regions involved in fear memory.

Pantazopoulos H, Dolatshad H, Davis FC - PLoS ONE (2011)

Bottom Line: These changes were accompanied by increased c-Fos expression at ZT0.In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group.The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Northeastern University, Boston, Massachusetts, United States of America. hantazo@mclean.harvard.edu

ABSTRACT
Evidence demonstrates that rodents learn to associate a foot shock with time of day, indicating the formation of a fear related time-stamp memory, even in the absence of a functioning SCN. In addition, mice acquire and retain fear memory better during the early day compared to the early night. This type of memory may be regulated by circadian pacemakers outside of the SCN. As a first step in testing the hypothesis that clock genes are involved in the formation of a time-stamp fear memory, we exposed one group of mice to fox feces derived odor (TMT) at ZT 0 and one group at ZT 12 for 4 successive days. A separate group with no exposure to TMT was also included as a control. Animals were sacrificed one day after the last exposure to TMT, and PER2 and c-Fos protein were quantified in the SCN, amygdala, hippocampus, and piriform cortex. Exposure to TMT had a strong effect at ZT 0, decreasing PER2 expression at this time point in most regions except the SCN, and reversing the normal rhythm of PER2 expression in the amygdala and piriform cortex. These changes were accompanied by increased c-Fos expression at ZT0. In contrast, exposure to TMT at ZT 12 abolished the rhythm of PER2 expression in the amygdala. In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group. TMT exposure at either time point did not affect PER2 or c-Fos in the SCN, indicating that under a light-dark cycle, the SCN rhythm is stable in the presence of repeated exposure to a fear-inducing stimulus. Taken together, these results indicate that entrainment to a fear-inducing stimulus leads to changes in PER2 and c-Fos expression that are detected 24 hours following the last exposure to TMT, indicating entrainment of endogenous oscillators in these regions. The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus.

Show MeSH

Related in: MedlinePlus

Schedule of TMT exposure.After 10 days of baseline wheel running activity in a LD cycle, one group of mice were exposed to TMT for 4 days at ZT 0 and to a control KimWipe with no odor at ZT 12 (shaded gray area) (A). Animals were then given one full 24 hour cycle without exposure before being sacrificed at 6 hour intervals on the following cycle. A second group of mice were exposed to a control KimWipe at ZT 0 and to TMT at ZT 12 (B).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105109&req=5

pone-0020658-g001: Schedule of TMT exposure.After 10 days of baseline wheel running activity in a LD cycle, one group of mice were exposed to TMT for 4 days at ZT 0 and to a control KimWipe with no odor at ZT 12 (shaded gray area) (A). Animals were then given one full 24 hour cycle without exposure before being sacrificed at 6 hour intervals on the following cycle. A second group of mice were exposed to a control KimWipe at ZT 0 and to TMT at ZT 12 (B).

Mentions: Adult male C3H mice (36) and male C57BL6 mice (12) were purchased from Charles River Breeding Laboratories (Wilmington, MA). Animals were maintained in a 12∶12 hour light dark (LD) cycle in light-tight black boxes with food and water available ad libitum. ZT 0 was designated as lights on, and ZT 12 was designated as lights off. All experiments were performed according to protocols approved by Northeastern University's Internal Animal Care and Use Committee (protocol # 10-0102 R). One group of C3H (12 mice) was exposed to fox feces derived 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) (Contech Inc., Delta, British Columbia) at ZT 0 for 4 successive days (TMT0 group) (Figure 1) by removing the cages from the light tight boxes and transferring them to an adjacent room where a KimWipe with 10 µl of TMT was held above the cage of each animal for 10 minutes. TMT has been established to elicit unconditioned (innate) fear response in rodents [39], [40], [41], [42], [43]. The TMT 0 group was also removed from the light-tight boxes at ZT 12 and brought to the adjacent room where a KimWipe without TMT was held above their cages for 10 minutes as a negative control in order to control for effects of disturbance to the animals at this time point. Lighting conditions in the adjacent room corresponded to the lighting conditions of the animals ZT time, thus animals were never exposed to light outside of the 12∶12 LD cycle. Animals were brought into the adjacent room for exposure to TMT or an empty KimWipe during the light's on portion of the LD cycle, thus the first group, receiving exposure to an empty sterile KimWipe, was brought out 20 minutes before lights off or 20 minutes after lights on, and the second group receiving TMT exposure was brought out 10 minutes before lights off or 10 minutes after lights on. Although animals were disturbed at the time point during which they do not receive TMT, they were not removed from their cages and thus were not introduced to a separate environment. It is important to control for disturbances to animals when analyzing effects related to circadian rhythm, as repeated disturbances to animals may affect circadian rhythm, c-Fos, or clock protein expression regardless of the type of stimulus (fear-inducing vs non fear-inducing). A second group of 12 C3H mice was exposed to TMT at ZT12 (TMT 12) for four successive days and exposed to a negative control KimWipe in the same manner at ZT 0. The group of animals that did not receive TMT at a particular time point was always brought into the adjacent room first, as not to expose the animals to any TMT possibly remaining in the air. The adjacent room was properly ventilated to ensure that TMT was removed from the environment well before the 12 hours after which animals were brought into the room again. This negative control group was used to ensure that any differences observed in Per2 or c-Fos expression would be due to the effect of TMT exposure and not due to general disturbance of the animal. A third group of 12 C3H mice housed in the same conditions but not exposed to TMT and not disturbed by being brought to the adjacent room was included in the study in order to have a group of normal control animals with no disturbances or TMT exposure as an absolute baseline control group.


A fear-inducing odor alters PER2 and c-Fos expression in brain regions involved in fear memory.

Pantazopoulos H, Dolatshad H, Davis FC - PLoS ONE (2011)

Schedule of TMT exposure.After 10 days of baseline wheel running activity in a LD cycle, one group of mice were exposed to TMT for 4 days at ZT 0 and to a control KimWipe with no odor at ZT 12 (shaded gray area) (A). Animals were then given one full 24 hour cycle without exposure before being sacrificed at 6 hour intervals on the following cycle. A second group of mice were exposed to a control KimWipe at ZT 0 and to TMT at ZT 12 (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105109&req=5

pone-0020658-g001: Schedule of TMT exposure.After 10 days of baseline wheel running activity in a LD cycle, one group of mice were exposed to TMT for 4 days at ZT 0 and to a control KimWipe with no odor at ZT 12 (shaded gray area) (A). Animals were then given one full 24 hour cycle without exposure before being sacrificed at 6 hour intervals on the following cycle. A second group of mice were exposed to a control KimWipe at ZT 0 and to TMT at ZT 12 (B).
Mentions: Adult male C3H mice (36) and male C57BL6 mice (12) were purchased from Charles River Breeding Laboratories (Wilmington, MA). Animals were maintained in a 12∶12 hour light dark (LD) cycle in light-tight black boxes with food and water available ad libitum. ZT 0 was designated as lights on, and ZT 12 was designated as lights off. All experiments were performed according to protocols approved by Northeastern University's Internal Animal Care and Use Committee (protocol # 10-0102 R). One group of C3H (12 mice) was exposed to fox feces derived 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) (Contech Inc., Delta, British Columbia) at ZT 0 for 4 successive days (TMT0 group) (Figure 1) by removing the cages from the light tight boxes and transferring them to an adjacent room where a KimWipe with 10 µl of TMT was held above the cage of each animal for 10 minutes. TMT has been established to elicit unconditioned (innate) fear response in rodents [39], [40], [41], [42], [43]. The TMT 0 group was also removed from the light-tight boxes at ZT 12 and brought to the adjacent room where a KimWipe without TMT was held above their cages for 10 minutes as a negative control in order to control for effects of disturbance to the animals at this time point. Lighting conditions in the adjacent room corresponded to the lighting conditions of the animals ZT time, thus animals were never exposed to light outside of the 12∶12 LD cycle. Animals were brought into the adjacent room for exposure to TMT or an empty KimWipe during the light's on portion of the LD cycle, thus the first group, receiving exposure to an empty sterile KimWipe, was brought out 20 minutes before lights off or 20 minutes after lights on, and the second group receiving TMT exposure was brought out 10 minutes before lights off or 10 minutes after lights on. Although animals were disturbed at the time point during which they do not receive TMT, they were not removed from their cages and thus were not introduced to a separate environment. It is important to control for disturbances to animals when analyzing effects related to circadian rhythm, as repeated disturbances to animals may affect circadian rhythm, c-Fos, or clock protein expression regardless of the type of stimulus (fear-inducing vs non fear-inducing). A second group of 12 C3H mice was exposed to TMT at ZT12 (TMT 12) for four successive days and exposed to a negative control KimWipe in the same manner at ZT 0. The group of animals that did not receive TMT at a particular time point was always brought into the adjacent room first, as not to expose the animals to any TMT possibly remaining in the air. The adjacent room was properly ventilated to ensure that TMT was removed from the environment well before the 12 hours after which animals were brought into the room again. This negative control group was used to ensure that any differences observed in Per2 or c-Fos expression would be due to the effect of TMT exposure and not due to general disturbance of the animal. A third group of 12 C3H mice housed in the same conditions but not exposed to TMT and not disturbed by being brought to the adjacent room was included in the study in order to have a group of normal control animals with no disturbances or TMT exposure as an absolute baseline control group.

Bottom Line: These changes were accompanied by increased c-Fos expression at ZT0.In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group.The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Northeastern University, Boston, Massachusetts, United States of America. hantazo@mclean.harvard.edu

ABSTRACT
Evidence demonstrates that rodents learn to associate a foot shock with time of day, indicating the formation of a fear related time-stamp memory, even in the absence of a functioning SCN. In addition, mice acquire and retain fear memory better during the early day compared to the early night. This type of memory may be regulated by circadian pacemakers outside of the SCN. As a first step in testing the hypothesis that clock genes are involved in the formation of a time-stamp fear memory, we exposed one group of mice to fox feces derived odor (TMT) at ZT 0 and one group at ZT 12 for 4 successive days. A separate group with no exposure to TMT was also included as a control. Animals were sacrificed one day after the last exposure to TMT, and PER2 and c-Fos protein were quantified in the SCN, amygdala, hippocampus, and piriform cortex. Exposure to TMT had a strong effect at ZT 0, decreasing PER2 expression at this time point in most regions except the SCN, and reversing the normal rhythm of PER2 expression in the amygdala and piriform cortex. These changes were accompanied by increased c-Fos expression at ZT0. In contrast, exposure to TMT at ZT 12 abolished the rhythm of PER2 expression in the amygdala. In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group. TMT exposure at either time point did not affect PER2 or c-Fos in the SCN, indicating that under a light-dark cycle, the SCN rhythm is stable in the presence of repeated exposure to a fear-inducing stimulus. Taken together, these results indicate that entrainment to a fear-inducing stimulus leads to changes in PER2 and c-Fos expression that are detected 24 hours following the last exposure to TMT, indicating entrainment of endogenous oscillators in these regions. The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus.

Show MeSH
Related in: MedlinePlus