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Sexually divergent expression of active and passive conditioned fear responses in rats

  1. Tina M Gruene
  2. Katelyn Flick
  3. Alexis Stefano
  4. Stephen D Shea
  5. Rebecca M Shansky Is a corresponding author
  1. Northeastern University, United States
  2. Cold Spring Harbor Laboratories, United States
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Cite as: eLife 2015;4:e11352 doi: 10.7554/eLife.11352

Abstract

Traditional rodent models of Pavlovian fear conditioning assess the strength of learning by quantifying freezing responses. However, sole reliance on this measure includes the de facto assumption that any locomotor activity reflects an absence of fear. Consequently, alternative expressions of associative learning are rarely considered. Here we identify a novel, active fear response (‘darting’) that occurs primarily in female rats. In females, darting exhibits the characteristics of a learned fear behavior, appearing during the CS period as conditioning proceeds and disappearing from the CS period during extinction. This finding motivates a reinterpretation of rodent fear conditioning studies, particularly in females, and it suggests that conditioned fear behavior is more diverse than previously appreciated. Moreover, rats that darted during initial fear conditioning exhibited lower freezing during the second day of extinction testing, suggesting that females employ distinct and adaptive fear response strategies that improve long-term outcomes.

https://doi.org/10.7554/eLife.11352.001

eLife digest

Animals can respond to fear in a variety of ways, such as by standing still (freezing), or rapidly escaping from an apparent threat. Freezing is the most common measure of fear that has been used in research studies. However, since the vast majority of these experiments have used male animals, it is not clear if freezing is a sufficient measure of fear in females.

To address this question, Gruene et al. analyzed different types of fear responses in large groups of male and female rats. The experiments used a technique called cued fear conditioning, which pairs a sound with a mild electrical shock to a foot. When rats learn that the sound predicts the shock, the sound alone causes them to produce a fear response. However, if the sound is then played repeatedly without a footshock, the rats learn to become less fearful of the sound in another learning process called “extinction”.

The experiments found that females were four times more likely than males to display fear in the form of rapid movements (referred to as “darting”). Animals that displayed darting were also less likely to freeze in response to the sound cue, which suggests that darting may represent an alternative fear strategy that is more common in females. During a subsequent extinction test, females that darted also displayed quicker reductions in both types of fear responses, which suggests that darting might be an active coping response that promotes long term reductions in fear.

Gruene et al.’s findings suggest that there are differences in the ways that males and females respond in fear of a threatening stimulus, and highlight the importance of analyzing a variety of fear responses in experiments. The next steps will be to understand the biological basis of the darting response in female rats.

https://doi.org/10.7554/eLife.11352.002

Introduction

In the laboratory, auditory or “cued” fear conditioning and extinction in rodents are the predominant tools for studying the neural mechanisms of learning and memory for aversive stimuli (Blanchard and Blanchard, 1969; LeDoux, 2000; Maren, 2001). In these assays, the strength of a tone-shock association is traditionally measured by the fraction of time during the conditioned stimulus (CS) that subjects exhibit freezing, defined as the cessation of all movement not required for respiration (Fanselow, 1980). Accordingly, low freezing is generally interpreted as reflecting a weak association and thus poor learning. Likewise, low freezing after extinction is taken to indicate successful suppression of the conditioned response, a new memory (Quirk and Mueller, 2008). However, by their construction, these traditional assays are insensitive to alternative expressions of fear, such as escape.

Most studies of fear conditioning and extinction in rodents use exclusively male subjects (Lebron-Milad and Milad, 2012). The few studies that directly compare conditioned freezing responses in males and females produced mixed results (Shansky, 2015) but most frequently reported lower freezing in females (Gupta et al., 2001; Maren et al., 1994; Pryce et al., 1999). Whether this effect reflects genuine learning deficits in females, or is related to sex differences in fear response strategies is unknown. For example, females reliably exhibit heightened ambulation in a wide variety of common behavioral tests (Archer, 1975; Fernandes et al., 1999; File, 2001; Seney et al., 2012), which may influence their selection of responses to threatening stimuli.

To identify possible alternative fear response strategies, we evaluated locomotor activity in gonadally intact adult male (n=56) and female (n=58) Sprague Dawley rats as they were trained and tested in auditory fear conditioning (5 habituation CS followed by 7 CS-US pairs), extinction (20 CS), and extinction retention (3 CS) tests across 3 days(Gruene et al., 2015) (Figure 1a). In many animals, we qualitatively observed a rapid ‘darting’ behavior during fear conditioning tone presentation–a rapid, forward movement across the chamber that resembled an escape-like response (illustrated in Figure 1b, and Video 1). We quantified these responses by identifying and counting them as discrete events in traces of each animal’s velocity for all sessions using Noldus Ethovision software and custom Matlab code (see source code 1, Materials and methods, and Figure 1c). We calculated darting rate (darts/min) during four non-overlapping trial epochs: 1) 60 s pre-CS period, 2) 30 s CS presentation, 3) 5 s “shock response” period, and 4) 30 s post-shock period (Figure 2a). This approach allowed us first to determine if darting reflected an alternate conditioned response, and second, whether the expression of conditioned darting predicted distinct behavioral patterns across fear conditioning and extinction.

Darting is an active learned response to the CS that occurs primarily in female rats.

(a) Experimental timeline. (b) Darts were characterized by a brief, high velocity movement across the test chamber. (c) Velocity traces from a representative animal, demonstrating increase in conditioned darting events across fear conditioning trials. Asterisks denote events that reached criterion for darting during the CS. Time 0 denotes CS onset. (d) Temporal organization of darting in all female rats. On the left is a two dimensional histogram of dart timing relative to the CS averaged over all females for 5 habituation trials and 7 conditioning trials on day 1. Trial time is on the x-axis and colored bars denote the trial epochs we defined as CS (green), shock response (orange), and post shock response (blue). Each row represents a CS trial (habituation 1–5, and conditioning 6–12), and depicts average dart rate by the color in each 4-second bin according to the color bar. On the right are histograms of the temporal organization of darts averaged over the five habituation trials (top) and the last three conditioning trials (bottom). Darts were detected and counted as described in Materials and methods. (e) Temporal organization of darting in all male rats. Panels are organized as in (d). During habituation trials, darts occurred at low rates throughout the trial in both sexes. In contrast, after conditioning only females exhibited increased darting triggered by tone onset (‘CS’) and sustained darting after shock delivery (‘Post-shock’). Both sexes darted in response to the shock itself (Shock response). In both sexes, the first bin after the shock exceeds the limits of the y-axis.

https://doi.org/10.7554/eLife.11352.003
Video 1
Example of conditioned darting.

A “Darter” during CS 12, corresponding to the last trace in Figure 1C. CS onset begins at 0:02 and continues through the entirety of the 18 sec video. The word “DART” appears on screen in red text during each of three observable darts.

https://doi.org/10.7554/eLife.11352.004

Results and Discussion

Prior to the initiation of shocks, darts were not temporally structured with respect to the CS. However, we found that females, but not males, exhibited increased dart frequency in response to CS onset during late trials (Figure 1c–e), suggesting that darting is a learned response. Figure 1d,e represent dart frequency amongst entire female and male cohorts, respectively.

Sex differences in darting responses during fear conditioning and extinction.

(a) The 4 fear conditioning epochs in which velocity was recorded. Graphs in c-f and i-j are color coded to match, and represent mean +/- SEM. (b) In graphs c-f and i-j, females are represented by filled circle, males by an open square. (c) Pre-CS (final 60 sec before 1st CS presentation) and CS dart rate (darts/min) during conditioning. (d) number of darts observed during 5s shock (US) response periods. (e) maximum velocity reached during 5s shock (US) response periods. (f) mean dart rate observed during 30s post-shock period. (g) and (h) Pearson’s correlations of mean shock response velocity and total session dart count [note that visible male outlier was removed from analysis for being 6 SDs above mean total dart count. When included, r=0.34, p<0.05]. (i) Pre-CS and CS dart rate (darts/min) during Extinction. (j) Pre-CS and CS dart rate (darts/min) during Extinction testing. *p<0.05; **p<0.01; *** p<0.001; ****p<0.0001 males vs. females.

https://doi.org/10.7554/eLife.11352.005

We next compared darting in males and females across all test sessions. Females exhibited higher CS dart rates than males on all 3 days (Figure 2c,i,j; conditioning: ns p=0.07, Mann Whitney test. p<0.001 2-way ANOVA main effect of sex, F1,112=12.1; sex x trial interaction F11,1232=2.12, p=0.02 Extinction: p=0.01, Mann Whitney test. p<0.05, 2-way ANOVA main effect of sex, F1,112=4.05. Extinction test: p=0.008, Mann Whitney test (Pre-CS); p<0.001, 2-way ANOVA main effect of sex, F1,112=14.58 (CS)). Notably, CS dart rate in females increased as CS-US presentations progressed (Figure 2c) and decreased during extinction (Figure 2i), again suggesting that darting may reflect an alternate expression of associative learning. During fear conditioning, although both males and females reliably darted during the shock response period (Figure 2d; p<0.01 2-way ANOVA main effect of sex, F1,112=8.5), shock-evoked darts in females reached higher velocities than darts in males (Figure 2e; p<0.0001 2-way ANOVA main effect of sex, F1,112=20.35). Additionally, females were more likely to dart during the 30s post-shock period than males (Figure 2f; p<0.0001 2-way ANOVA main effect of sex, F1,112=23.27). To determine whether an animal’s shock response velocity was related to its overall propensity to dart, we correlated the mean velocity reached across all 7 US presentations with total detected darts during fear conditioning. These measures were significantly correlated in females (Figure 2g) but not males (Figure 2h), suggesting that in females only, an animal’s immediate reaction to an aversive stimulus may influence its future response strategies.

We did not observe darting in all females, however, and so to identify possible behavioral markers and outcomes of darting, we divided animals into ‘Darter’ and ‘Non-darter’ subgroups. An animal qualified as a “Darter” if it exhibited at least one dart during fear conditioning tones (CS) 8–12. CS 8 is the 3rd CS-US pairing, and the point at which we usually observe a robust increase in freezing in males. Therefore, only darts that occurred during this same time period were considered to reflect conditioned darting. Over 40% of females qualified as Darters (Figure 3a), whereas approximately 10% of males qualified (Figure 3f; chi-square = 13.8, p=0.0002). There was no effect of the estrous cycle on darting (Figure 3 - supplement). Compared to Non-darters, female Darters exhibited greater shock response velocities (Figure 3b; p=0.001 2-way ANOVA main effect of group F1,56=11.49), as well as higher dart rates in the post-shock period (Figure 3c; p<0.0001 2-way ANOVA main effect of group, F1,56=25.42.), suggesting that female Darters have a more robust and protracted response to the shock. Importantly, female Darters did not exhibit higher dart rates during pre-CS periods or during CS-only habituation trials (Figure 3d; p=0.65, Mann Whitney test), suggesting that Darters are not simply more active overall, and were not pre-disposed to dart in response to the CS. During the CS, Darters exhibited increased darting as CS-US pairs progressed (p<0.0001 2-way ANOVA group x trial interaction, F11,616=8.8; main effect of group F1,56=26.35, p<0.0001). During the Extinction Pre-CS period, Darters did not dart more than Non-darters (p=0.38 Mann Whitney), but Darters exhibited increased darting during the first Extinction CS, suggesting that darting is a conserved conditioned response (2-way ANOVA interaction, F19,1064=1.584, p=0.05; *p<0.05 Sidak’s post-hoc test).

Figure 3 with 1 supplement see all
Darting subpopulations are greater in females and exhibit distinct behavioral patterns.

(a) and (f) proportion of females and males that qualified as Darters. (b) max velocity reached during shock response period (c) mean dart rate (darts/min) observed during 30s post-shock period. (d) Pre-CS and CS dart rate (darts/min) during conditioning, extinction, and extinction test. (e) CS freezing in female Darters vs. Non-darters. (g) Shock response velocity did not differ between male Darters and Non-darters. (h) mean dart rate (darts/min) observed during 30s post-shock period. (i) Pre-CS and CS dart rate (darts/min) during conditioning, extinction, and extinction test. (j) CS freezing in male Darters vs. Non-darters. *p<0.05; **p<0.01; *** p<0.001; ****p<0.0001 Darters vs. Non-darters

https://doi.org/10.7554/eLife.11352.006

We next asked whether CS darting during fear conditioning related to CS freezing behavior (Figure 3e). In females, Darters and Non-darters did not differ in pre-CS or CS-only (habituation) freezing during fear conditioning. However, as CS-US pairings progressed, Darters froze less than Non-darters, suggesting that increased darting may prevent or compete with freezing responses p=0.02 2-way ANOVA group x trial interaction F11,616=2.16. main effect of darting F1,56=4.18, p<0.05). Darters and Non-darters did not significantly differ in freezing during Extinction. However, Darters also froze less during the extinction test (day 3; p<0.02, 2-way ANOVA main effect of group F1,56=5.76) despite not exhibiting increased darting at that time, suggesting that darting during fear conditioning does not simply compete with an animal’s freezing response, but may also reflect an adaptive response that predicts positive outcomes after extinction learning.

In the small subpopulation of male Darters, CS dart rate (Figure 3i; Conditioning p<0.0001, 2-way ANOVA group x trial interaction, F11,594=3.76. Extinction: p<0.0001, 2-way ANOVA group x trial interaction, F19,1026=3.17) and freezing (Figure 3j; Conditioning: p<0.01 2-way ANOVA group x trial interaction F11,583=2.68. Extinction and extinction test: No significant interaction or effects) patterns during fear conditioning shared some characteristics with those in females.

However, there are several notable distinctions between male and female Darters. First, CS dart rate in darting males was characterized by a steady low rate of darting across trials (Figure 3i), instead of the increase across trials observed in females (Figure 3d), suggesting that darting in males may not reflect a learned fear response, but general hyperactivity that results in less freezing. Second, unlike our observations in females, male Darters did not exhibit heightened shock response velocities (Figure 3g) or robust post-shock dart rates (Figure 3h; p=0.01 2-way ANOVA group x trial interaction, F6,324=2.8, no main effects) compared to Non-darters. Third, male Darters did not exhibit lower freezing during extinction testing, suggesting that the potential long-term behavioral implications of darting during fear conditioning are stronger in females than in males. Together with the large observed sex difference in darting prevalence (Figure 2a,f), these discrepancies suggest that there may be qualitative differences in the potential causes and effects of darting in males versus females. Further work will be necessary to determine whether the neurobiological basis of darting is comparable in males and females.

In summary, our data show that during auditory fear conditioning, a substantial subpopulation of predominantly female rats exhibit an active conditioned response associated with reduced conditioned freezing throughout fear conditioning and extinction tests. To our knowledge, this is the first formal characterization of conditioned escape-like responses during classical fear conditioning, in which the shock cannot be avoided. In contrast, learned escape behavior has been well studied in Active Avoidance (AA) paradigms (Galatzer-Levy et al., 2014; Martinez et al., 2013), and although research into potential sex differences in AA is rare, females are reported to learn AA faster than males (Dalla and Shors, 2009), which is consistent with females preferring active fear responses over freezing.

One potentially provocative finding here is that female Darters exhibited comparable freezing to Non-darters at the start of extinction, but enhanced extinction retention the following day. Importantly, lower freezing during extinction retention could not be explained by increased darting during this phase. This suggests that darting during fear conditioning does not interfere with the formation or memory of the tone-shock association, but may confer a long-term protective or adaptive state that promotes increased cognitive flexibility and thus enhanced extinction maintenance (Maren et al., 2013). This effect is reminiscent of reports from Maier and colleagues, who have convincingly demonstrated that perceived “escapability” in a shock stress paradigm leads to enhanced AA in subsequent testing (reviewed in Maier, 2015). In a similar vein, increases in “active coping” behavior (digging, rearing, wall-sniffing) during a cued fear memory test are positively correlated with AA success (Metna-Laurent et al., 2012). Recruitment of these active coping fear responses instead of freezing has been shown to involve neural transmission in the central amygdala (Gozzi et al., 2010) and depend on cannabinoid signaling (Metna-Laurent et al., 2012), but to date have not been studied in female rodents. Importantly, these responses have not been demonstrated during fear conditioning learning, the stage at which darting appears to be most critical. Clearly, a great deal of work remains to dissect the neurobiological mechanisms that mediate darting, and to determine its relevance to other indices of active coping, especially in female model organisms.

The finding that conditioned darting occurs primarily in females holds major implications for the interpretation of fear conditioning and extinction studies that use both male and female rats, suggesting that freezing alone may not be a complete measure of learned fear in female subjects. Specifically, female rats that exhibit low freezing levels during fear conditioning could be erroneously described as expressing low fear and/or poor learning, when in fact they have engaged darting responses. This phenomenon may also be clinically relevant, pointing to a sex-specific threat response that predicts enhanced extinction maintenance. Because the learning processes that underlie extinction form the basis for exposure therapy (a common treatment for Post-Traumatic Stress Disorder [PTSD]), a better understanding of the mechanisms that drive darting could lead to improved exposure therapy success. Women are at a twofold risk for PTSD compared to men, and thus identification of the neurobiological factors that determine darting in females may provide insight into sex differences in coping strategies, as well as in stress susceptibility and resilience.

Materials and methods

Subjects

Young adult (8–10 weeks) male (n=56) and female (n=58) Sprague Dawley rats were individually housed in the Nightingale Animal Facility at Northeastern University on a 12:12 light:dark cycle with access to food and water ad libitum. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Northeastern University Institutional Animal Care And Use Committee. All experimenters were female.

Estrous cycle monitoring

Females were vaginally swabbed daily for two weeks to ensure normal estrous cycling. Collected cells were smeared on a microscope slide, stained with Nissl, and examined with a light microscope for cytology.

Behavioral testing

Apparatus and stimuli

Rats underwent habituation, fear conditioning and fear extinction as in (Gruene et al, 2015) in one of four identical chambers constructed of aluminum and Plexiglas walls (Rat Test Cage, Coulbourn Instruments, Allentown, PA), with metal stainless steel rod flooring that was attached to a shock generator (Model H13–15; Coulbourn Instruments). The chambers were lit with a single house light, and each chamber was enclosed within a sound-isolation cubicle (Model H10–24A; Coulbourn Instruments). An infrared digital camera allowed videotaping during behavioral procedures. Chamber grid floors, trays, and walls were thoroughly cleaned with water and dried between sessions. Rats were allowed to freely explore the chamber for 4 min before tone presentation on each day began.

Fear conditioning

After a 4-minute acclimation period, all rats were exposed to five tone (CS) presentations (habituation), followed by seven conditioning trials (CS–US pairings) on day 1. The CS was a 30-s, 5 kHz, 80 dB SPL sine wave tone, which co-terminated with a 0.5-s, 0.7 mA footshock US during fear conditioning. Mean intertrial interval was 4 min (2–6 min range) throughout habituation and fear conditioning. Freezing was continuously recorded during the conditioning session and analyzed using FreezeFrame Software. Minimum bout was set at 2sec. After conditioning, rats were returned to their home cages.

Extinction

Freezing was recorded continuously during the extinction training (20 CS presentations, day 2) and test sessions (3 CS presentations, day 3). Both extinction training and testing took place in the same chamber as fear conditioning, but with different contextual cues (floor, light, and odor). Mean inter-trial interval was 4min (2–6 min range).

Locomotor activity analysis

Video files from FreezeFrame were extracted as QuickTime File Format (.mov) and then converted to MPEG-2 files using AVS Video Converter 9.1 (Online Media Technologies LTD. 2014). The MPEG-2 files were then run through EthoVision software (Noldus), with a center point tracking with a velocity sampling rate of 3.75. Velocity data were computed by Noldus software at 3.75 Hz sampling rate and exported to Matlab (Mathworks). Darts were detected in the exported trace using the findpeaks function with a minimum velocity of 23.5 cm/s and a minimum interpeak interval of 0.8 s. The 23.5 cm/s threshold for darts was determined by cross-referencing velocity data with experimenter scoring of darting behavior. 23.5 cm/s was the velocity at which all movements at that rate or higher were consistently scored as darts. These discrete events were registered to each trial and analyzed with custom Matlab software (available as a source code 1 file).

Statistical analysis

Darting, velocity, and freezing values during each epoch were averaged for each group and analyzed for each session (fear conditioning, extinction, extinction test) using 2-way repeated measure ANOVAs with factors of group and trial. Mann-Whitney t-tests were used for all Pre-CS comparisons. One male animal was removed from analysis because its total dart count was 6 standard deviations outside the mean (shown in Figure 2h).

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Decision letter

  1. Peggy Mason
    Reviewing Editor; University of Chicago, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled "Sexually divergent expression of active and passive conditioned fear responses in rats" for consideration by eLife. Your article has been favorably evaluated by a Senior Editor and four peer reviewers, one of whom, Peggy Mason, is a member of our Board of Reviewing Editors. One the four reviewers has agreed to reveal her identity: Margaret McCarthy.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

All reviewers believe this is an important and timely topic that has the potential to greatly advance our understanding of hitherto contradictory results and ultimately help to accurately illuminate the effects of fear on behavior. It was broadly felt that the information provided will be of substantial use to researchers in the U.S. who are required (by NIH) to incorporate sex as a biological variable in all preclinical research.

Essential revisions:

1) Better define darting and darters. What constitutes darting? How much darting at what time points makes a rat into a darter? Justify the criteria chosen for defining darters. Are the male darters really darters or just fast-moving dart-impostors that do not show the full phenotype that female darters show? The graphs suggest that the male darters are quite different from the female ones.

2) The design of the study is a bit unusual. There are 5 CS alone presentations prior to conditioning aimed at measuring habituation to the CS. However, this design induces latent inhibition slowing down the acquisition of the CS-US association. However, the interpretation by the authors that a lack of darting during the CS-only trials indicates that darting responses reflect conditioning is a bit problematic. The authors should also explain the unusual design with 5 CS presentations as well as the choice to not use unpaired CS-US presentations as controls (the typical approach).

3) A longer Discussion is needed to pick up the points raised throughout the Results but never discussed (e.g. the suggestion that darting is an adaptive response linked to positive outcomes and not simply competition for shared motor resources).

4) Work by Gozzi et al. (2010) and Laurent and colleagues (2012) demonstrate that conditioned freezing behaviors are inhibited to favor active coping strategies in classical fear conditioning paradigms. Thus, the temporal decrease in freezing behaviors observed in both male and female rats during extinction should be accompanied by an increase in active coping behaviors in these experimental animals. Did the authors not observe any switch from passive to active behaviors, and vice versa, in these experimental animals? Were other active coping strategies not present in male mice that were distinct from the darting phenotype present in female rats? Did female mice not exhibit other forms of active coping strategies other than darting? Alternatively, if the authors are arguing that their observed "darting" is different form of fear behavior than that of active or passive, it would serve the authors well to reconcile these claims with the current literature. In sum, please place the present results into the context of active vs passive coping strategies, adaptive strategies, and why those may differ between males and females.

5) Examine whether there are individual differences in darting that may be a function of weight or any available measure of locomotion such as open field. A number of additional specific suggestions would strengthen the manuscript. As one example, a specific comparison of freezing and darting across trials would be of interest (see below).

6) Could the authors include a movie of darting?

Minor Comments:

Reviewer #1:

Given the reports of the effect of male hormones (from experimenters) on rodent behavior (e.g. Mogil work), please specify the gender of the experimenters.

Please explicitly state that Figure 1 and Figure 2 include all males/females (not just the darters), if that is the case. In any case clarify.

Please clarify the following passage: "Compared to non-darters, female darters exhibited greater shock response velocities […] as well as higher dart rates in the post-shock period." Darters are defined by their darting rates during the post-shock period. If the reviewer is correct, remove this. If the reviewer is wrong, please explain why.

Reviewer #2:

1) One can't help notice that the darting behavior by females learning to fear the stimulus sounds remarkably similar to the "hopping and darting" that sexually receptive females show to solicit male attention. Can the authors comment on this and do they have any way of comparing these two darting behaviors to see if they are in fact similar or distinct?

2) Wouldn't you expect dart velocity to be related to body weight? And is that why females have a higher velocity?

Reviewer #3:

It would be useful to compare the darting behavior to freezing behavior more directly in Figure 1. Specifically, it would be helpful to see similar graphics for Figure 1c, d, and e for freezing behavior to visualize the similarity and differences in darting and freezing across training trials.

It would be interesting to know if previous studies have found that males and females have more similar acquisition rates for conditioned suppression (where a decrease in bar pressing for a food reward is a measure of fear) than for fear conditioning. If so, that would further support the argument that there are better measures of fear in females.

Reviewer #4:

In Figure 1, female rats exhibit greater darts/min as compared to male rats in control conditions (CS only) and prior to fear conditioning. Though the authors argue that there are no within-sex differences in dart rates between darters and non-darters prior to conditioning, it would serve the authors well to examine whether there are locomotor differences between female and male cohorts prior to fear conditioning. This will help the authors to determine whether pre-existing activity differences predispose rats to exhibiting a darting phenotype as a learned fear behavior.

To strengthen their argument, the authors should not only compare dart rate extinction between female and male rats but they should also compare the freezing behaviors for both male and female rats as well (Figure 2i and j).

In Figure 3i, there are no significant differences in the dart rate during extinction training (20CS) yet significance is indicated (****).

For the supplemental data, please indicate the statistic test that was carried out to "determine there was no effect of the estrous cycle on darting”.

https://doi.org/10.7554/eLife.11352.012

Author response

Essential revisions:

1) Better define darting and darters. What constitutes darting? How much darting at what time points makes a rat into a darter? Justify the criteria chosen for defining darters.

In our response to this set of questions, we feel it is important to distinguish between our definition of a dart (as a discrete event), a darter in the narrow sense (an individual that was included in our darting analysis group) and a darter in the broad sense (a ‘true darter’ as opposed to an ‘imposter’). We have crisply defined the first two entities, however it was not our goal to delineate true darting. We completely agree with the reviewer that this is a very interesting question, but we simply note in the Discussion that there are crucial features to the darting exhibited by females that are not seen in males. The implication is that male darting, as observed in this study, is qualitatively different from female darting, however we did not attempt to define or segregate true darters.

We have now expanded the text of the manuscript to more clearly define the criteria we used to define both darting and Darters (Results and Discussion, as well as in “Locomotor activity analysis” section of Materials and methods). In addition, we include a video (Video 1) of an example of darting. In order to be classified as a Darter, an animal must have exhibited at least one dart during fear conditioning CS (tone presentation) 8-12. Animals that darted in response to shock or during inter-trial intervals but not during the CS were not considered Darters. Because darting during fear conditioning has not been previously characterized, we chose to set a relatively inclusive criterion. As we move forward with our work and begin to dissect the neurobiological basis of darting, we expect that the criteria may be refined.

Are the male darters really darters or just fast-moving dart-impostors that do not show the full phenotype that female darters show? The graphs suggest that the male darters are quite different from the female ones.

Although the male Darters reach the criterion of one dart during late fear conditioning CS presentations, we agree that they do not exhibit many of the other characteristics we observe in female Darters (as discussed above). These behavioral differences between male and female Darters are discussed in paragraph six of Results and Discussion, but clearly further work is needed to determine the functional significance, if any, of darting in males.

2) The design of the study is a bit unusual. There are 5 CS alone presentations prior to conditioning aimed at measuring habituation to the CS. However, this design induces latent inhibition slowing down the acquisition of the CS-US association.

This is an interesting and important point. Although latent inhibition can be induced by prior exposure to a CS, we do not feel that it is a significant risk with our particular preparation. To date, latent inhibition in fear conditioned rats has only been demonstrated when CS pre-exposure is considerable and CS-US pairings brief, e.g. a pre-exposure of 20 or 30 tone presentations prior to a single CS-US presentation (De la Casa, 2013; Rudy, 1994), when tone pre-exposure is protracted in duration (Baker et al., 2012), or when it occurs over several sessions (Sotty et al., 1996). In other words, it appears that in order for latent inhibition to occur in cued fear conditioning, CS pre-exposure must be far more robust than 5 30-sec CSs, especially given the fairly high number of CS-US pairings (7) in our preparation. Importantly, we have used this design in three recent publications (Gruene et al., 2014, 2015; Rey et al., 2014) and reliably observe rapid increases in freezing to CS during fear conditioning learning and testing, suggesting that latent inhibition plays a minimal, if any, role in our behavioral outcomes.

However, the interpretation by the authors that a lack of darting during the CS-only trials indicates that darting responses reflect conditioning is a bit problematic.

We apologize if our interpretation of the lack of CS-only darting was unclear or overstated. While those data alone cannot indicate whether darting is a conditioned response, we infer that at the very least, darting is not an unconditioned response to the tone, i.e. there is nothing inherent about the tone that elicits darting in Darters, nor are Darters simply “jumpy” animals. We have added to the manuscript text (paragraph three, Results and Discussion) to clarify this point. However, we feel that the most convincing evidence that darting is a conditioned response is the fact that CS-specific darting can be observed in female Darters at the start of extinction (Figure 3d), when the animal does not experience any footshocks. This response is predicted by the presence of darting during conditioning. It is also important to note that during conditioning, female Darters show increased propensity to dart specifically during the tone. We argue that these observations collectively constitute strong evidence that darting is a conditioned response.

The authors should also explain the unusual design with 5 CS presentations as well as the choice to not use unpaired CS-US presentations as controls (the typical approach).

This design was based on numerous publications from experts in the field (Burgos-Robles et al., 2007; Bush et al., 2007; Galatzer-Levy et al., 2013; Milad and Quirk, 2002; Sotres-Bayon et al., 2007), and is fairly common for studies in which extinction of cued fear is a focus.

We want to emphasize that the 5 CS habituation tones were not intended to represent a “control” condition, but merely a way of establishing baseline responsiveness to the CS prior to its association with the US. We agree that a thorough examination of darting in alternate CS-US arrangements will be an important next step in defining the situational boundaries of darting. In an unpaired CS-US design, the CS would signal safety (Pollak et al., 2010), and thus we would not expect to observe increased darting to the CS as we do in a paired design here. But because of the large scale of the current study, the novelty of the sex-specific findings within, and the topical nature of these findings with respect to recent changes in NIH policy, we felt it was appropriate to report the current findings before undertaking another large-scale study.

3) A longer Discussion is needed to pick up the points raised throughout the Results but never discussed (e.g. the suggestion that darting is an adaptive response linked to positive outcomes and not simply competition for shared motor resources).

We have now expanded the Discussion to elaborate on these points (please see paragraph eight).

4) Work by Gozzi et al. (2010) and Laurent and colleagues (2012) demonstrate that conditioned freezing behaviors are inhibited to favor active coping strategies in classical fear conditioning paradigms. Thus, the temporal decrease in freezing behaviors observed in both male and female rats during extinction should be accompanied by an increase in active coping behaviors in these experimental animals. Did the authors not observe any switch from passive to active behaviors, and vice versa, in these experimental animals?

We have read both of these excellent papers carefully, and while on the surface they appear to be relevant to the current study because they also deal with active and passive behaviors, there are considerable distinctions in design and behavioral measures that make direct extrapolation to our work difficult. During extinction, an animal learns not to fear the CS. We would therefore expect all forms of fear responses (both passive and active) to subside. This is indeed what we observe, as can be seen in Figure 3. It’s worth noting that in both referenced papers, animals underwent a comparably weak conditioning procedure – either “partial” conditioning (Gozzi et al., 2010) or a single CS-US pairing (Metna Laurent et al., 2012) vs. our 7 CS-US pairings – and were tested for fear memory/extinction during a single 6- or 8-minute CS exposure, vs. our 90 minute, 20 CS extinction session. It is likely that the differences in CS and US parameters influenced the behavioral strategies and trajectories animals engaged in those studies and ours. That said, we would predict that had the extinction sessions in both papers been substantially longer, they would have also observed a decrease in active responding.

Were other active coping strategies not present in male mice that were distinct from the darting phenotype present in female rats? Did female mice not exhibit other forms of active coping strategies other than darting? Alternatively, if the authors are arguing that their observed "darting" is different form of fear behavior than that of active or passive, it would serve the authors well to reconcile these claims with the current literature.

Active coping behaviors in both papers were similarly defined as “digging, exploration, and rearing,” or “digging, rearing, and wall-sniffing,” none of which would have qualified as darting. Because the specific goal of the current manuscript is to define darting as a conditioned fear response, we did not evaluate these other active coping behaviors (some, such as digging, were not possible because there was no bedding in the test chambers). However, a thorough characterization of all measurable behaviors during fear conditioning and extinction in the future will undoubtedly be informative.

It is also important to note that the test phase in which behavioral analysis was focused is different between these papers and the current study. In both Gozzi et al and Metna-Laurent et al, behaviors were measured only during the fear memory/extinction phase (i.e. Day 2), and behavior during fear conditioning itself is not reported. In contrast, the key darting phase in our analysis is fear conditioning (Day 1) – Darters are defined based on darting behavior during this phase, and our hypothesis is that the engagement of darting during fear conditioning elicits long term changes that influence behavior on subsequent test days. We look forward to testing this hypothesis in follow-up studies.

Finally, subjects in the referenced studies were exclusively male mice, so it is not known whether their observations would have carried to female mice, let alone female rats, as were used in the current study.

In sum, please place the present results into the context of active vs passive coping strategies, adaptive strategies, and why those may differ between males and females.

We have added to the Discussion of the manuscript to address the relevance of our findings to these papers and others (paragraph eight).

5) Examine whether there are individual differences in darting that may be a function of weight or any available measure of locomotion such as open field.

We have provided data to address these issues below, in response to Minor Points from Reviewer 2 (weight) and Reviewer 4 (locomotion).

6) Could the authors include a movie of darting?

We now include a movie of darting (Video 1).

Minor Comments:

Reviewer #1:

Given the reports of the effect of male hormones (from experimenters) on rodent behavior (e.g. Mogil work), please specify the gender of the experimenters.

We now provide this information in the Materials and methods, under “subjects.” All experimenters were female.

Please explicitly state that Figure 1 and Figure 2 include all males/females (not just the darters), if that is the case. In any case clarify.

We now explicitly state this in the manuscript text, in addition to the Figure caption. All 114 animals (58 females, 56 males) are represented in all figures. It is only in Figure 3 that animals are separated into “Darters” and “Non-darters.”

Please clarify the following passage: "Compared to non-darters, female darters exhibited greater shock response velocities […] as well as higher dart rates in the post-shock period." Darters are defined by their darting rates during the post-shock period. If the reviewer is correct, remove this. If the reviewer is wrong, please explain why.

We apologize that the criteria for Darting were unclear. Darters were categorized exclusively based on whether they darted during the CS (tone presentation), not on their dart rate during the postshock period, in which there is no CS. We then evaluated shock response velocities and postshock dart rate in these groups (Figure 3b and 3c). As noted above in our response to comment 1, we have expanded our explanation of both darting and Darter criteria and hope this clarifies the issue.

Reviewer #2:

1) One can't help notice that the darting behavior by females learning to fear the stimulus sounds remarkably similar to the "hopping and darting" that sexually receptive females show to solicit male attention. Can the authors comment on this and do they have any way of comparing these two darting behaviors to see if they are in fact similar or distinct?

This is an interesting question. On a strictly locomotor basis, the darts we observe here are fairly similar in appearance to those during solicitation. It is of course not yet known whether the neural mechanisms that elicit darting in these two vastly different scenarios overlap. However, we do not observe any “hopping” behaviors that resemble those of solicitation behavior (Erskine, 1989). Moreover, we observe conditioned darting in females of all estrous stages (see Figure 3-figure supplement 1) as well as in a few males, suggesting that conditioned darting is not simply indicative of female proceptivity. There is also no aspect of the task that could be expected to elicit sexual behaviors in females (e.g. there is no male or male pheromones present during the behavior).

2)Wouldn't you expect dart velocity to be related to body weight? And is that why females have a higher velocity?

It makes sense that the lighter an animal is, the more easily it could move around, thus making it more likely to dart. To address the possibility that lower weight predisposed animals to darting, we compared body weight between Darters and Non-Darters, but found no significant differences (Author response image 1). Although males clearly weigh more than females, we feel these data suggest that weight alone cannot account for the incidence of darting.

Reviewer #3:

It would be useful to compare the darting behavior to freezing behavior more directly in Figure 1. Specifically, it would be helpful to see similar graphics for Figure 1c, d, and e for freezing behavior to visualize the similarity and differences in darting and freezing across training trials.

We agree that comparing darting and freezing side-by-side would be informative. However, the visual representations of darting in Figure 1 depict event frequencies for male and female populations at given time points. Because freezing is not a discrete event, it does not lend itself to this form of representations. We feel that the best comparison of freezing and darting can be observed in Figure 3, which intentionally shows darting and freezing directly above one another. In this way, you can see that Darters’ and Non-darters’ trajectories of darting and freezing are inverse.

It would be interesting to know if previous studies have found that males and females have more similar acquisition rates for conditioned suppression (where a decrease in bar pressing for a food reward is a measure of fear) than for fear conditioning. If so, that would further support the argument that there are better measures of fear in females.

To date, there is very little existing data on sex differences in conditioned suppression. However, Maes (2002) did not find a sex difference in the magnitude of conditioned fear suppression. It is worth noting that in Active Avoidance studies, females have shown enhanced learning compared to males (Dalla and Shors, 2009). We now include these studies in the Discussion.

Reviewer #4:

In Figure 1, female rats exhibit greater darts/min as compared to male rats in control conditions (CS only) and prior to fear conditioning. Though the authors argue that there are no within-sex differences in dart rates between darters and non-darters prior to conditioning, it would serve the authors well to examine whether there are locomotor differences between female and male cohorts prior to fear conditioning. This will help the authors to determine whether pre-existing activity differences predispose rats to exhibiting a darting phenotype as a learned fear behavior.

As we note in the second paragraph of the manuscript Introduction, there is a vast literature demonstrating heightened locomotor activity in females compared to males. When we examine baseline pre-CS locomotor activity (measured as mean velocity in cm/s), we also observe higher mean velocities in females ( Author response image 2, p<0.0001, unpaired t-test). However, baseline locomotor activity was not different between Darters and Non-darters in either sex, suggesting that locomotor activity does not predict the likelihood of darting.

To strengthen their argument, the authors should not only compare dart rate extinction between female and male rats but they should also compare the freezing behaviors for both male and female rats as well (Figure 2i and j).

We have previously compared freezing during extinction in males and females, and find no overall sex differences in extinction (Rey et Al., 2014; Gruene et al., 2015). Although general sex differences in freezing during extinction was not the focus of the current manuscript, we have provided those data below. We find a slight but significant main effect of sex in freezing extinction learning (F1,112=4.02, p=0.048, 2-way repeated measures ANOVA). We believe the discrepancy between this result and our previous findings is due to the considerable statistical power conferred by n’s of ~60/sex. We do not observe an effect of sex in extinction retrieval (F1,112=1.84, p=0.18, 2-way repeated measures ANOVA). Data are represented in Author response image 3.

In Figure 3i, there are no significant differences in the dart rate during extinction training (20CS) yet significance is indicated (****).

We have fixed this error.

For the supplemental data, please indicate the statistic test that was carried out to "determine there was no effect of the estrous cycle on darting”.

We ran a chi-square test to identify differences in the estrous cycle distribution between Darters and Non-darters, which returned a value of 2.785, p=0.42. This information has been added to the manuscript.

https://doi.org/10.7554/eLife.11352.013

Article and author information

Author details

  1. Tina M Gruene

    1. Department of Psychology, Northeastern University, Boston, United States
    Contribution
    TMG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Katelyn Flick

    1. Department of Psychology, Northeastern University, Boston, United States
    Contribution
    KF, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  3. Alexis Stefano

    1. Department of Psychology, Northeastern University, Boston, United States
    Contribution
    AS, Acquisition of data, Contributed unpublished essential data or reagents, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  4. Stephen D Shea

    1. Cold Spring Harbor Laboratories, Cold Spring Harbor, United States
    Contribution
    SDS, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0001-6927-0189
  5. Rebecca M Shansky

    1. Department of Psychology, Northeastern University, Boston, United States
    Contribution
    RMS, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    1. r.shansky@neu.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Institute of Mental Health (R21 MH098006-01)

  • Rebecca M Shansky

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

 We thank Michael Gunson, Jesse Katon, Marc Lowe, and Heather Brenhouse for technical support, and Mark Baxter for manuscript comments.

Ethics

Animal experimentation: All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Northeastern University Institutional Animal Care and Use Committee protocol # 12-0102R.

Reviewing Editor

  1. Peggy Mason, Reviewing Editor, University of Chicago, United States

Publication history

  1. Received: September 3, 2015
  2. Accepted: November 12, 2015
  3. Accepted Manuscript published: November 14, 2015 (version 1)
  4. Version of Record published: December 23, 2015 (version 2)

Copyright

© 2015, Gruene et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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