Fig 1.
Males and females can both indirectly acquire fear memories.
(A) Experimental design. The observer rats watch the same sex non-cage mate demonstrator rats undergo auditory fear conditioning in Context A. After two days, the observer is placed back into the training context for 5 minutes without receiving any foot shock or auditory cues and immediately transferred into Context B to be tested for fear to the auditory cue. (B) Behavioral performance of the observer during the training session. Both males and females show increased freezing after the first shock presentation to the demonstrator, though females showed less freezing behavior overall than males (minutes 4–9). (C) Behavioral performance of the observer during the training context retention test. (D) Memory retention of the observer during (CS) and between (post-CS) auditory cue presentations during the test session. Both sexes exhibited increased freezing behavior in response to the tone. Testing is shown as a bin of 3 CS or post-CS periods. N = 8 per group. Data show as mean with SEM. * Denotes p < 0.05 from females.
Fig 2.
Sex of the demonstrator does not affect the acquisition of indirectly acquired fear memories.
(A) Male and female observer rats were paired with a demonstrator of either the same or opposite sex. (B-C) Changing the sex of the demonstrator did not affect the observer’s performance in either sex for retention to the context (B) or auditory cue (C). Auditory cue testing is shown as a bin of 3 CS or post-CS periods. N = 7–8 per group. Data show as mean with SEM. F = female, M = male, O = observer, D = demonstrator.
Fig 3.
Novelty of the demonstrator does not affect the acquisition of indirectly acquired fear memories.
(A) Male and female observer rats were paired with a demonstrator that was either a cage mate (familiar) or non-cage mate (novel) rat of the same sex. Changing the novelty of the demonstrator did not affect the observer’s performance in either sex during the testing session for the context (B) or auditory cue (C). Auditory cue testing is shown as a bin of 3 CS or post-CS periods. N = 8 per group. Data show as mean with SEM. F = female, M = male, CM = cage mate.
Fig 4.
Indirectly acquired fear responses are behaviorally similar to those of pseudoconditioning.
Rats were either trained with a novel same-sex demonstrator (observer) or without any demonstrators present in the partitioned chamber (pseudoconditioned) and then tested for the freezing response in context B two days later. (A-B) No significant differences were observed between the two groups during the context test (A) or during (CS) or between (post-CS) auditory cue presentations (B) during the test session. N = 6 per group, all males. Data show as mean with SEM.
Fig 5.
Indirect fear memories have unique, sex-specific protein degradation changes in the amygdala following training.
(A) Male and female rats were trained in an indirect fear conditioning procedure that included a demonstrator (received tone and shock) and an observer (received tone while watching demonstrator). Pseudoconditioned animals were placed on the observer side of the chamber and presented with the tone without a demonstrator present. One hour after their respective training session, the basolateral amygdala (BLA) was collected, purified with K48-polyubiquitin Tandem Ubiquitin Binding Entity (TUBE) and analyzed with liquid chromatography mass spectrometry. All groups were compared to behaviorally naïve animals of the same sex with N = 6 per group per sex. (B-C) Heat maps showing the log2 value of proteins with significantly altered levels of K48-polyubiquitination (differentially ubiquitinated proteins; DUPs) in females (B) and males (C) in different training groups compared to naïve animals. White bars indicate that protein was not identified as significantly altered in that group.
Fig 6.
Indirect fear memories have unique, sex-specific protein degradation changes in the anterior cingulate cortex following training.
Male and female rats were trained in an indirect fear conditioning procedure that included a demonstrator (received tone and shock) and an observer (received tone while watching demonstrator). Pseudoconditioned animals were placed on the observer side of the chamber and presented with the tone without a demonstrator present. One hour after their respective training session, the anterior cingulate cortex (ACC) was collected, purified with K48-polyubiquitin Tandem Ubiquitin Binding Entity (TUBE) and analyzed with liquid chromatography mass spectrometry. All groups were compared to behaviorally naïve animals of the same sex with N = 6 per group per sex. (A-B) Heat maps showing the log2 value of proteins with significantly altered levels of K48-polyubiquitination (differentially polyubiquitinated proteins; DUPs) in females (A) and males (B) in different groups compared to naïve animals. White bars indicate that protein was not identified as significantly altered in that group.
Fig 7.
Indirectly acquired fear memories engage immediate early gene expression patterns in the retrosplenial cortex that are similar to directly acquired fear memories but distinct from pseudoconditioning.
RNA was collected from the retrosplenial cortex from animals described above in Figs 4 and 5. Gene expression levels of two memory-related immediate early genes¸Egr2 and c-fos, were measured by RT-qPCR. (A-B) Egr2 mRNA levels were significantly higher in observer animals than pseudoconditioned animals in both females (A) and males (B), though did not differ from demonstrator animals in either sex. (C) c-fos mRNA levels were significantly higher in observer animals than pseudoconditioned animals in females, though did not differ from demonstrator animals. N = 5 per group in females, and N = 6–8 per group in males. Data show as mean with SEM. NS: non-significant. * Denotes p < 0.05 from Naïve. # Denotes p < 0.05 from Demonstrator. $ Denotes p < 0.05 from Observer.