Behavioral and neural correlates of diverse conditioned fear responses in male and female rats

Abstract

Pavlovian fear conditioning is a widely used tool that models associative learning in rodents. For decades the field has used predominantly male rodents and focused on a sole conditioned fear response: freezing. However, recent work from our lab and others has identified darting as a female-biased conditioned response, characterized by an escape-like movement across a fear conditioning chamber. It is also accompanied by a behavioral phenotype: Darters reliably show decreased freezing compared to Non-darters and males and reach higher velocities in response to the foot shock ("shock response"). However, the relationship between shock response and conditioned darting is not known. This study investigated if this link is due to differences in general processing of aversive stimuli between Darters, Non-darters and males. Across a variety of modalities, including corticosterone measures, the acoustic startle test, and sensitivity to thermal pain, Darters were found not to be more reactive or sensitive to aversive stimuli, and, in some cases, they appear less reactive to Non-darters and males. Analyses of cFos activity in regions involved in pain and fear processing following fear conditioning identified discrete patterns of expression among Darters, Non-darters, and males exposed to low and high intensity foot shocks. The results from these studies further our understanding of the differences between Darters, Non-darters and males and highlight the importance of studying individual differences in fear conditioning as indicators of fear state.

Figure 1.. Fear conditioning induces comparable increases in corticosterone levels in all animals.

A. Experimental design consisted of blood plasma samples taken immediately before and 15 minutes after a 7 CS-US fear conditioning paradigm. B. Corticosterone levels increased in fear conditioned males and females, but not in CS-only males and females. C. Corticosterone levels increased in all Darters and Non-darters following fear conditioning. (**) p < 0.01, (***) p < 0.001, (****) p < 0.00001. Design figure in panel A created in Birorender.

Figure 2.. Darters do not exhibit enhanced startle responses.

A. Experimental design consisted of the acoustic startle test where animals were exposed to a series of startle-evoking white noises at four different decibel levels presented in a random order for one hour. One week later, animals ran through a 7 CS-US fear conditioning paradigm. B. Darter females have a greater latency to startle than non-darter females and males at 95 db stimuli. C. Males have a greater peak startle amplitude than darter females at 105 db and 110 db stimuli. (*) p < 0.05, (**) p < 0.01, (****) p < 0.00001. Design figure in panel A created in Birorender.

Figure 3.. Behavior results for Experiment 3.

A. Experimental design consisted of one day of the hot plate assay to obtain baseline thermal pain threshold. One week following exposure to the hot plate, animals ran through a 7 CS-US fear conditioning paradigm in one of three groups: 1mA shock, 0.3mA shock, or CS-only control groups. B. Proportion of Darters in males and females across 0.3 and 1 mA shock intensity groups and heatmaps for both sexes showing prevalence of conditioned darting across fear conditioning tone C. Female Non-darters have a longer latency to withdraw their hind paw than males. D. Female Darters have a longer latency to withdraw their forepaw than males. E. Darters consistently withdraw their hind paws before their forepaws, a pattern not seen in Males or non-Darters. F. Percent of time spent freezing increased because of increasing shock intensity in males G. Percent of time spent freezing increased with higher shock intensity in females. H. In females, non-Darters froze more than Darters, regardless of shock intensity. I. Shock response increased with higher shock intensity in males J. Shock response increased with higher shock intensity in females. K. In Females, Darters showed higher shock response than non-Darters exposed to the same shock intensity. (*) p < 0.05, (**) p < 0.01. Design figure in panel A created in Birorender.

Figure 4.. cFos activity in the dorsal horn of the lumbar spine following fear conditioning.

A. Experimental design for cFos portion of the experiment. B. Representative image of L3 of the Dorsal Horn of the Lumbar stained with DAB for cFos. C. There were no differences in cFos expression across shock intensities or sexes. D. There were no differences in cFos expression across conditioned responses. Dotted line indicates CS-only animal cFos levels. Design figure in panel A created in Birorender.

Figure 5.. cFos activity in the lateral parabrachial nucleus following fear conditioning.

A. representative image of the lPbN following staining. B. There was as significant effect of shock in females only, and a significant effect of sex, driven by the 1 mA animals. C. When females were separated by conditioned response, 1 mA animals trended towards higher cFos levels, regardless of CR. (*) p < 0.05. Dotted line indicates CS-only animal cFos levels.

Figure 6.. cFos analyses from the columns of the periaqueductal gray following fear conditioning.

A. Representative image of the immunofluorescent staining for cFos in the PAG. Image has been edited for this figure to make cells more visible to the naked eye. B-C. cfos+ cells in the dmPAG, by sex and for females only by conditioned response. No differences were found between any groups. D. There was a significant effect of sex in the dlPAG: Females had more cFos expression than males. E. There was no significant effect of shock or conditioned response in the dlPAG. F. No sex or shock effect was found in the lPAG. G. There was a significant effect of conditioned response in the lPAG: Darters showed increased cFos+ cells in the lPAG compared to Non-darters, regardless of shock intensity. H. There was no effect of shock or sex in the vlPAG. I. There was no effect of conditioned response in the vlPAG. (*) p < 0.05. Dotted line indicates CS-only animal cFos levels.

Figure 7.. cFos analyses from the lateral, central, and basolateral amygdala.

A. Representative image of DAB cFos staining in the lateral amygdala. −2.76 from Bregma. B. There was no main effect of shock intensity or sex in the lateral amygdala. C. There was a significant effect of shock intensity and a significant interaction between conditioned response and shock intensity in females: At higher shock intensities, female Darters had more cFos expression in the lateral amygdala than Non-darters. D. Representative image of DAB cFos staining in the central amygdala. −2.40 from Bregma. E. No effects of shock intensity or sex were found in the central amygdala. F. There was a significant shock x conditioned response interaction in females: Female Non-darters had significantly less cFos expression in the central amygdala at higher shock intensities; Darter did not follow this pattern. G. Representative image of DAB cFos staining in the basolateral amygdala. −3.00 from Bregma. H. There was a significant effect of shock intensity in the BLA, although post-hoc tests did not reach significance. I. There was a significant shock x conditioned response interaction in females: Non-darters decreased cFos expression at higher shock intensities while Darters increased. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Dotted line indicates CS-only animal cFos levels.

Figure 8.. Correlations of cFos expression following fear conditioning of the analyzed regions.

A. Correlations between males exposed to 0.3 mA shock and 1 mA shock. B. Correlations between females exposed to 0.3 mA shock and 1 mA shock. Significant positive correlations shown with yellow connecting lines and significant negative correlations shown with blue connecting line. If N’s for any correlation were less than 10, lines are gray. Solid gray lines indicate positive correlation. Dashed gray lines indicate negative correlation. Level of significance is indicated by the thickness of the line.