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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Neurobiol Learn Mem. 2021 Aug 13;184:107501. doi: 10.1016/j.nlm.2021.107501

Genetic background determines behavioral responses during fear conditioning

LR Seemiller 1,*, SM Mooney-Leber 2,*, E Henry 1, A Hess 1, A Druffner 1, G Peltz 3, TJ Gould 1
PMCID: PMC8572373  NIHMSID: NIHMS1734393  PMID: 34400349

Abstract

Freezing behavior is used as a measure of a rodent’s ability to learn during fear conditioning. However, it is possible that the expression of other behaviors may compete with freezing, particularly in rodent populations that have not been thoroughly studied in this context. Rearing and grooming are complex behaviors that are frequently exhibited by mice during fear conditioning. Both behaviors have been shown to be stress-sensitive, and the expression of these behaviors is dependent upon strain background. To better understand how genetic background impacts behavioral responses during fear conditioning, we examined freezing, rearing, and grooming frequencies prior to fear conditioning training and across different stages of fear conditioning testing in male mice from eight inbred mouse strains (C57BL/6J, DBA/2J, FVB/NJ, SWR/J, BTBR T+ ltpr3Tf/J, SM/J, LP/J, 129S1/SvlmJ) that exhibited diverse freezing responses. We found that genetic background determined rearing and grooming expression throughout fear conditioning, and their patterns of expression across stages of fear conditioning were strain dependent. Using publicly available SNP data, we found that polymorphisms in Dab1, a gene that is implicated in both grooming and learning phenotypes, separated the strains with high contextual grooming from the others using a hierarchical clustering analysis. This suggested a potential genetic mechanism for the observed behavioral differences. These findings demonstrate that genetic background determines behavioral responses during fear conditioning and suggest that shared genetic substrates underlie fear conditioning behaviors.

Keywords: Fear conditioning, genetics, behavior, freezing, rearing, grooming

1. INTRODUCTION

Fear conditioning is used to study learned associations between aversive and neutral stimuli (Maren, 2001; Pavlov, 1927). Subjects can be trained to associate an aversive, unconditioned stimulus with a context or discrete conditioned stimulus (i.e., fear conditioning). The size of the fear response during testing is used as an index of fear learning, where a greater fear response indicates stronger fear learning. This can be used to measure fear learning processes that are conserved between humans and rodents. Freezing, or the absence of non-respiratory movement, is a well-established fear behavior that is assessed in rodent fear conditioning studies (Fanselow, 1980, 1994). While this analysis method has been thoroughly validated in prior research, many prior studies examine populations with limited genetic diversity.

Comparisons of inbred mouse strains can reveal genetic contributions to phenotypes. They have been used to study genetic differences in a wide range of learning paradigms (Bolivar et al., 2001; Delprato et al., 2015; Nie & Abel, 2001; O’Leary et al., 2011; Owen et al., 1997; Park et al., 2011). Analysis of inbred strain panels has led to successful identification of genetic loci associated with learning (Wehner et al., 1997). However, studies examining complex behaviors such as learning must consider how strain differences in lower order processes might affect performance when higher order learning tasks are examined. For example, strain differences in visual ability can account for learning differences observed in the Morris water maze and Barnes maze tests (Brown & Wong, 2007; O’Leary et al., 2011). Hence, it is reasonable to expect that behavioral differences across inbred strains may compete with the expression of freezing (a traditional measure of fear learning) and thus, with the interpretation of fear learning abilities. It is also possible that rodent expression of fear learning might involve conditioned behaviors other than freezing.

Rearing and grooming behaviors are both frequently expressed throughout fear conditioning (Fitch et al., 2002; Tipps et al., 2014) and their expression in an open field arena varies across genetic backgrounds (Delprato et al., 2017). Rearing is an exploratory behavior that has been associated with both locomotion and stress or fear responses (Crusio & van Abeelen, 1986; Crusio, 2001). Prior studies have described complex relationships between exploratory behavior, perception of novelty, and spatial learning. Such studies have found genetic associations of hippocampal mossy fiber morphology with exploratory rearing behavior (Crusio et al., 1989) and spatial learning ability (Delprato et al., 2015). Grooming is a stress-sensitive, highly stereotyped self-maintenance behavior (Kalueff et al., 2016; Sturman et al., 2018; Veloso et al., 2016). Stress-induced patterns of grooming may follow an inverted U-shaped curve, with high- and low-stress conditions suppressing grooming behavior and mildly stressful conditions increasing grooming behavior (Fernández-Teruel & Estanislau, 2016). However, patterns of grooming may be more nuanced, as grooming bouts and duration can be influenced by grooming microstructure, or the pattern of grooming movements, which also changes in response to stress (Kalueff et al., 2007; Song et al., 2016). Because rearing and grooming phenotypes vary across strains and are susceptible to stress effects, we were interested in how they were expressed during fear conditioning in a genetically diverse population.

It has been suggested that freezing may not be the only quantifiable expression of fear during fear conditioning testing (Fitch et al., 2002). Evidence suggests that female rats are more likely to express active fear responses (darting) than male rats during cued fear conditioning (Gruene et al., 2015). Others have examined rearing and grooming behaviors as possible expressions of fear during fear conditioning testing. These behaviors can be modulated by stress (Fernández-Teruel & Estanislau, 2016; Sturman et al., 2018; Veloso et al., 2016) and are expressed during fear conditioning training and testing (Fitch et al., 2002; Tipps et al., 2014). Differences in the expression of rearing and grooming throughout different stages of fear conditioning have been reported in C57BL/6J and DBA/2J mice (Fitch et al., 2002; Tipps et al., 2014). It has also been reported that a mouse genetic model of Tourette syndrome demonstrates enhanced grooming in response to a tone after cued fear conditioning (Xu et al., 2015). These findings could suggest that there are genetic differences in 1) The expression of fear (as freezing, grooming, or rearing), 2) The expression of rearing and grooming that interfere with the expression of fear (as freezing), or 3) The expression of rearing and grooming that do not affect the expression of fear (as freezing).

To further examine these possibilities, we quantified rearing and grooming frequencies prior to fear conditioning training and during fear conditioning testing across male mice from eight inbred strains. These strains were selected because we observed them demonstrating a wide range of freezing responses during fear conditioning. To better understand how fear conditioning affects rearing and grooming, difference scores (comparing unconditioned behaviors to conditioned behaviors) were calculated across stages of fear conditioning. Behaviors were correlated across conditioning stages and with publicly available phenotypes to better understand shared genetic contributions and potential confounding phenotypes. Finally, a clustering analysis utilizing publicly available genetic data was used to assess the relationship between a gene of interest, Dab1, and grooming and learning phenotypes.

2. MATERIALS AND METHODS

2.1. Subjects

Subjects were adult (10–15 weeks old) male mice from eight inbred strains [C57BL/6J, DBA/2J, FVB/NJ, SWR/J, BTBR T+ ltpr3Tf/J (BTBR), SM/J, LP/J, 129S1/SvlmJ (129S1)] ordered from Jackson Laboratory (Bar Harbor, ME). Mice were group-housed, given ad libitum access to food and water, and kept on a 12-hour light/dark cycle. All fear conditioning occurred during the light phase, between 8:00 AM and 5:00 PM. All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Penn State Institutional Animal Care and Use Committee. Sample sizes were 9–10 per strain.

Subjects in the described study were saline-treated control subjects in a larger study examining strain differences in drug responses. Strains were selected for this study based on observed differences in freezing responses during fear conditioning. Specifically, when compared to all strains surveyed in the larger strain panel, FVB/NJ, SWR/J, and BTBR strains showed low contextual freezing, C57BL/6J and DBA/2J strains showed average contextual freezing, and 129S1, SM/J, and LP/J strains showed high contextual freezing (Fig 1A).

Figure 1.

Figure 1.

Figure 1.

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Freezing percentage across all four stages of fear conditioning (A) and conditioned changes in freezing represented as a percent change between either Baseline and Context and PreCS and CS (B) are determined by genetic background. n = 9–10 per strain. Data are shown as mean +/− SEM. * represents p < 0.05.

2.2. Saline Treatment

Mice received chronic saline treatment prior to fear conditioning because they were saline-treated control subjects in a larger study. Osmotic mini pumps (model #1002, Alzet Inc.; Cupertino, CA) were surgically implanted and administered saline subcutaneously for 12 days, as described previously (Davis et al., 2005; Goldberg et al., 2021; Portugal et al., 2012). Mini pump implantation and removal surgeries were performed using aseptic procedures and 2–3% isoflurane anesthesia.

2.3. Fear Conditioning

Because mice were control subjects in a larger study examining learning during nicotine withdrawal, mice were trained in fear conditioning one day after mini pumps were removed, as described previously (Davis et al., 2005; Goldberg et al., 2021; Portugal et al., 2012). Mice were trained and tested in noise-attenuating fear conditioning chambers (18.8×20×18.3 cm; MED Associates, St. Albans, VT) with a 65 dB background noise produced by chamber fans. Subjects were placed in conditioning chambers and monitored for 120 seconds (Baseline period). After the Baseline period, fear conditioning training consisted of two conditioned stimulus (CS; 30 seconds, 85-dB white noise)-unconditioned stimulus (US; 2 seconds, 0.45-mA footshock) pairings with a 120-second intertrial interval. The US was presented during the last two seconds of the CS. One day after training, subjects were returned to the training context and monitored for 300 seconds (Context period). Later that day, subjects were moved to a new conditioning chamber with different tactile and visual cues and monitored for 180 seconds without the conditioned stimulus (PreCS period) and 180 seconds with the conditioned stimulus tone (CS period).

2.4. Behavior Quantification

Freezing behavior, or the absence of movement aside from respiration, was quantified by EthoVision XT (Noldus, Wageningen, Netherlands). To quantify rearing and grooming behaviors, two observers blinded to conditions used a time-sampling method. Observations were recorded every ten seconds and divided by the total number of possible observations to determine rearing and grooming frequency (percentage) during each trial. Behavioral scores were spot-checked by two different observers also blinded to conditions. Rearing was defined as subjects lifting both paws into the air or placing both paws on chamber walls (Tipps et al. 2014; Bevins & Besheer 2006). Grooming was defined as subjects licking, rubbing, or scratching the paws, head, or body (Tipps et al. 2014; Kalueff & Tuohimaa 2004). Freezing, rearing, and grooming behavior frequencies were collected across Baseline, Context, PreCS, and CS periods. Cumulative behavior frequencies and behavior frequencies per minute were analyzed to account for possible time-dependent effects.

2.5. Correlations Across Phenotypes or Observation Periods

To assess potential genetic overlap in fear conditioned behaviors, strain means (freezing, rearing, and grooming) were correlated between behaviors within Context and CS periods. Additionally, to assess the role of conditioning in behavioral responses, strain means of the same behavior types (freezing, rearing, or grooming) were correlated across all stages of fear conditioning (Baseline, Context, PreCS, and CS). All comparisons were Pearson correlations.

2.6. Correlations with Publicly Available Phenotypes

Strain means for freezing, rearing, and grooming were compared using Pearson correlations with publicly available phenotypes from Mouse Phenome Database (MPD) (Bogue et al., 2018). Phenotypes were selected from trait lists of locomotor (MPD VT:0001392), anxiety-related (MPD VT:0010716), and learning/memory/conditioning (MPD VT:0002063) phenotypes. Data sets were only included if they contained five of our eight strains and at least one high-freezing, one average-freezing, and one low-freezing strain (according to freezing categories listed in Subjects section). Phenotypes using non-traditional assays were excluded, as this exploratory analysis intended to determine a relationship with conventional measurements. One representative phenotype was chosen per observation period within a study to avoid over-representation of redundant phenotypes within single experiments.

2.7. Statistical Analysis

Statistical analysis was conducted using SPSS 26 (IBM, Armonk, NY). One-way (Strain) ANOVA was used to examine the influence of Strain on freezing, rearing, and grooming within Baseline, Context, PreCS, and CS periods or on difference scores calculated across observation periods (Context behavior minus Baseline behavior, or CS behavior minus PreCS behavior). Within each subject, difference scores were calculated by subtracting Baseline or PreCS behavior frequencies from Context or CS behavior frequencies, respectively. Bonferroni-corrected planned comparisons (nonparametric one-sample sign tests) determined whether difference scores were significantly different from zero.

Mixed-model two-way (Strain, Timepoint) ANOVA was used to examine potential interactions between genetic background and timepoint on behavior frequencies. When Strain and Timepoint interactions were detected, Bonferroni-corrected planned comparisons (nonparametric sign tests) were used to compare behavior frequencies across timepoints within strains. When Strain and Timepoint interactions were detected in Baseline or Context periods, the first and last minutes of each period were compared within Strain. Because the PreCS and CS periods each contained three one-minute time bins, the last PreCS time bin (minute 3) and the first CS time bin (minute 4) were compared when interactions were detected to assess strain-specific responses to the introduction of the CS. Statistical significance threshold was set at Bonferroni-corrected p<0.05.

2.8. Hierarchical clustering analysis of SNPs

Hierarchical clustering analysis of Dab1 SNPs was also conducted in SPSS (IBM, Armonk, NY). Dissimilarity scores were calculated as counts of SNPs between all possible strain pairings. Complete Linkage/Furthest Neighbor methods were used to cluster strains by dissimilarity scores. SNP data were obtained from Mouse Phenome Database data set CGD-MDA1 which utilized the Mouse Diversity array to assess genetic variation among strains (Bogue et al., 2018; Yang et al., 2011).

3. RESULTS

3.1. Strain determines freezing behavior during fear conditioning

One-way (Strain) ANOVA was used to assess the impact of genetic background on freezing behavior during fear conditioning. Our analysis found a main effect of Strain on freezing frequency in Baseline (F7,68=22.14, p<0.001), Context (F7,68=84.99, p<0.001), PreCS (F7,68=18.28, p<0.001), and CS (F7,68=22.54, p<0.001) periods (Fig 1A), suggesting that strain determines freezing behavior across all stages of fear conditioning.

Two-way (Strain, Timepoint) ANOVA was used to assess possible timepoint effects on freezing behavior within fear conditioning periods (Supplemental Figs 1–3A). A two-way (Strain, Timepoint) ANOVA of freezing in the Baseline period identified a main effect of Strain (F7,68=22.13, p<0.001). In the Context period, we found main effects of Strain (F7,68=84.10, p<0.001) and Timepoint (F4,240=7.45, p<0.001) and a Strain by Timepoint interaction (F4,240=3.28, p<0.001). Sign tests did not find any significant differences between Context Minutes 1 and 5, the first and last minutes of the test, within strains. Across PreCS and CS periods, main effects of Strain (F7,68=27.53, p<0.001) and Timepoint (F3,219=218.32, p<0.001) and a Strain by Timepoint interaction (F23,219=5.55, p<0.001) were detected. Between Minutes 3 and 4 of the combined PreCS and CS periods (immediately before and after introduction of the CS), FVB/NJ (p=0.002), SWR/J (p=0.004), BTBR (p=0.004), C57BL/6J (p=0.002), and DBA/2J (p=0.002) strains significantly increased freezing behavior. The behavioral differences between Minutes 3 and 4 observed in some strains may indicate a behavioral response to the introduction of the CS.

To assess how conditioning changed the expression of fear conditioning behaviors, difference scores were calculated across Baseline and Context (Context – Baseline) or PreCS and CS (CS – PreCS) periods using changes in behavior frequencies of individual mice. One-way (Strain) ANOVA of freezing difference scores found a main effect of Strain on Context – Baseline (F7,68=72.26, p<0.001) and CS – PreCS (F7,68=8.87, p<0.001) difference scores. Planned comparisons (one-sample sign tests) found that all strains showed significant increases in freezing across Baseline and Context periods [FVB/NJ (p=0.002), SWR/J (p=0.004), BTBR (p=0.004), C57BL/6J (p=0.002), DBA/2J (p=0.002), 129S1 (p=0.002), SM/J (p=0.004), and LP/J (p=0.004)]. All strains except SM/J also showed increased freezing across PreCS and CS periods [FVB/NJ (p=0.002), SWR/J (p=0.004), BTBR (p=0.004), C57BL/6J (p=0.002), DBA/2J (p=0.002), 129S1 (p=0.002), and LP/J (p=0.004); (Fig 1B)]. Thus, most strains showed significant changes in freezing responses in both Context – Baseline and PreCS – CS difference scores.

3.2. Strain determines rearing behavior during fear conditioning

One-way (Strain) ANOVA found a main effect of Strain on rearing behavior during Baseline (F7,68=11.64, p<0.001), Context (F7,68=12.07, p<0.001), PreCS (F7,68=10.95, p<0.001), and CS (F7,68=12.32, p<0.001) periods (Fig 2A), suggesting that strain determines rearing behavior across all stages of fear conditioning.

Figure 2.

Figure 2.

Figure 2.

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Rearing percentage across all four stages of fear conditioning (A) and conditioned changes in rearing represented as a percent change between either Baseline and Context and PreCS and CS (B) are determined by genetic background. n = 9–10 per strain. Data are shown as mean +/− SEM. * represents p < 0.05.

Two-way (Strain, Timepoint) ANOVA assessed rearing behavior within fear conditioning stages (Supplemental Figs 1–3B). We found a main effect of Strain (F7,68=11.43, p<0.001) during the Baseline period. In the Context period, main effects of Strain (F7,68=12.19, p<0.001) and Timepoint (F3,208=8.25, p<0.001) were detected. Across combined PreCS and CS periods, there were main effects of Strain (F7,68=11.82, p<0.001) and Timepoint (F4,279=28.33, p<0.001) and a Strain by Timepoint interaction (F29,279=4.14, p<0.001). Within strain, planned comparisons (sign tests) of rearing frequencies found that only the SWR/J strain showed a significant change in rearing frequency between Minutes 3 and 4 (p=0.004). Thus, only the SWR/J strain showed a significant behavioral response to the introduction of the CS.

Difference scores were calculated to assess conditioning effects on rearing behavior. One-way (Strain) ANOVA examining rearing difference scores found a main effect of Strain in both Context - Baseline (F7,68=4.50, p<0.001) and CS - PreCS (F7,68=10.11, p<0.001) scores. Between Baseline and Context periods, planned comparisons (one-sample sign tests) found that SWR/J mice exhibited reduced rearing behavior (p=0.004). Between PreCS and CS periods, both SWR/J (p=0.004) and DBA/2J (p=0.002) strains had decreases in rearing behavior (Fig 2B). To summarize, SWR/J mice showed the most consistent decreases in rearing behavior in Context – Baseline and PreCS – CS difference scores.

3.3. Strain determines grooming behavior during fear conditioning

One-way (Strain) ANOVA was also used to determine strain effects on grooming behavior during fear conditioning. A main effect of Strain was found in Baseline (F7,68=4.24, p=0.001), Context (F7,68=23.52, p<0.001), PreCS (F7,68=7.81, p<0.001), and CS (F7,68=2.37, p=0.015) periods (Fig 3A), suggesting that strain determines grooming responses across all stages of fear conditioning.

Figure 3.

Figure 3.

Figure 3.

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Grooming percentage across all four stages of fear conditioning (A) and conditioned changes in grooming represented as a percent change between either Baseline and Context and PreCS and CS (B) are determined by genetic background. n = 9–10 per strain. Data are shown as mean +/− SEM. * represents p < 0.05.

Two-way (Strain, Timepoint) ANOVA was again used to assess grooming across observation periods (Supplemental Figs 1–3C). During the Baseline period, we found a main effect of Strain (F7,68=5.07, p<0.001) and a Strain by Timepoint interaction (F7,68=2.48, p=0.025). Post-hoc comparisons did not detect significant differences across Baseline timepoints within strains. In the Context period, main effects of Strain (F7,68=23.52, p<0.001) and Timepoint (F3,221=5.36, p=0.001) were detected. For combined PreCS and CS periods, main effects of Strain (F7,68=7.24, p<0.001) and Timepoint (F3,195=6.29, p=0.001) and a Strain by Timepoint interaction (F20,195=3.13, p<0.001) were detected. Within strain, planned comparisons found no significant differences in grooming frequencies between the time bins immediately before and after the CS (minutes 3 and 4). This suggests that there were not significant grooming responses to the introduction of the CS during testing.

One-way (Strain) ANOVA assessing grooming difference scores found a main effect of Strain in Context - Baseline (F7,68=9.56, p<0.001) and CS - PreCS (F7,68=5.46, p<0.001) scores. Between Baseline and Context periods, within-strain planned comparisons (one-sample sign tests) found that FVB/NJ mice significantly increased grooming behavior (p=0.002). No significant changes in grooming between PreCS and CS periods were detected within strain (Fig 3B). To summarize, grooming difference scores only detected a significant change in grooming behavior in the FVB/NJ strain between Baseline and Context periods.

3.4. Conditioned freezing negatively correlates with rearing and grooming

To evaluate the influence of shared genetic background on freezing, rearing, and grooming behaviors within observation periods, we correlated fear conditioning behaviors within Context and CS periods. In the Context period, freezing negatively correlated with both rearing (r=−0.776, p=0.023) and grooming (r=−0.736, p=0.037) behaviors. In the cued period, only rearing had a significant negative correlation with freezing behavior (r=−0.889, p=0.003; Table 1). Thus, freezing generally negatively correlated with rearing and grooming behaviors.

Table 1.

Pearson correlation coefficients between freezing, rearing, and grooming during both Context and CS periods. Bolded values represent significant correlations.

Context Correlations
Behavior Grooming Rearing
Rearing 0.27 --
Freezing −0.78 * −0.74 *
CS Correlations
Behavior Grooming Rearing
Rearing 0.24 --
Freezing −0.37 −0.89 **
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*

Correlation is significant at the 0.05 level;

**

Correlation is significant at the 0.01 level.

3.5. Rearing, grooming, and freezing behaviors positively correlate across stages of fear conditioning

To examine strain influence on freezing, rearing, and grooming across all stages of fear conditioning, strain means for each behavior were correlated across behavior observation periods (Table 2).

Table 2.

Pearson correlation coefficients for each behavior of interest (freezing, rearing, and grooming) across stages of fear conditioning. Bolded values represent significant correlations.

Freezing Correlations
Trial Baseline Context Pre-CS
Context 0.73 * -- --
Pre-CS 0.77 * 0.93 ** --
CS 0.40 0.58 0.72 *
Rearing Correlations
Trial Baseline Context Pre-CS
Context 0.86 ** -- --
Pre-CS 0.94 ** 0.74 * --
CS 0.54 0.66 0.48
Grooming Correlations
Trial Baseline Context Pre-CS
Context 0.49 -- --
Pre-CS 0.62 0.82 * --
CS 0.49 0.13 0.16
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*

Correlation is significant at the 0.05 level;

**

Correlation is significant at the 0.01 level.

Freezing in the PreCS period significantly correlated with freezing in all Baseline (r=0.769, p=0.026), Context (r=0.932, p=0.001), and CS (r=0.720, p=0.044) periods. Baseline freezing also correlated with Context freezing (r=0.730, p=0.040). Strain means for rearing during the Baseline period correlated with rearing during the Context (r=0.857, p=0.006) and PreCS (r=0.935, p=0.001) periods. Rearing during the Context period correlated with rearing during the PreCS period (r=0.742, p=0.035). Grooming behavior correlated between the Context and PreCS periods (r=0.819, p=0.013; Table 2). In summary, all behaviors correlated across stages of fear conditioning to some degree.

3.6. Publicly available locomotor, anxiety-like, and learning phenotypes correlate with fear conditioning behaviors

To assess the impact of Strain on fear conditioning behaviors, we correlated our data with publicly available locomotor, anxiety-like, and learning phenotypes from Mouse Phenome Database (MPD; phenome.jax.org; Bogue et al., 2018). A summary of significant correlations with publicly available learning phenotypes is shown in Table 3, and complete lists of learning, locomotor, and anxiety-related correlations are provided in Supplemental Tables 1–3. Correlations of fear conditioning behaviors with publicly available locomotor and anxiety-related phenotypes found some associations between behaviors, particularly between rearing and locomotor behaviors (Supplemental Tables 2–3).

Table 3.

Pearson correlation coefficients between behaviors collected during fear conditioning and other learning phenotypes (from publicly available data sets) across all conditions of fear conditioning. Bolded values represent significant correlations.

Behavior Learning Phenotype and MPD ID Baseline Context PreCS CS
Freezing Morris Water Maze acquisition training mean latency to find hidden platform, day 3 [s] 22541 (spatial learning) −0.68 −0.82 −0.97 ** −0.82
Rearing Barnes circular maze acquisition training mean errors (n) 22511 (spatial learning) −0.27 −0.01 −0.31 −0.99 **
Morris Water Maze acquisition training mean latency to find hidden platform, day 3 [s] 22541 (spatial learning) 0.96 * 0.88 0.97 ** 0.47
Grooming Morris water maze probe trial percent time, correct quadrant
(%) 22544 (spatial memory)		 0.90 * 0.27 0.50 0.63
Morris Water Maze acquisition training mean latency to find hidden platform, day 3 [s] 22541 (spatial learning) 0.31 0.88 * 0.80 −0.08
Morris Water Maze reversal training mean latency to find hidden platform, day 3 [s] 22542 (cognitive flexibility) 0.04 0.90 * 0.80 −0.26
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*

Correlation is significant at the 0.05 level;

**

Correlation is significant at the 0.01 level. O’Leary, Savoie, & Brown (2011) = 22511; Brown & Wong (2007) = 22511, 22544, & 2254.

Latency to find the hidden platform in a Morris Water Maze acquisition test (MPD 22541), where a greater latency indicates impaired spatial learning, negatively correlated with freezing in the PreCS period (r=−0.97, p<0.01), rearing in the Baseline (r=0.96, p=0.01) and PreCS (r=0.97, p=0.01) periods, and grooming during the Context period (r=0.88, p<0.05). Errors in a Barnes circular maze acquisition task (MPD 22511), where more errors indicate impaired spatial learning, correlated negatively with rearing during the CS period (r=−0.99, p<0.01). Percent time in the correct quadrant of a Morris Water Maze during the probe trial, where a greater amount of time is associated with better spatial memory, was positively associated with grooming during the Baseline period (MPD 22544; r=0.90, p=0.04). Latency to find the hidden platform during Morris Water Maze reversal training, where a greater latency represents impaired cognitive flexibility, was positively correlated with grooming during the Context period (MPD 22542; r=0.90, p=0.04; Brown & Wong, 2007; O’Leary et al., 2011).

3.7. Polymorphisms in Dab1 segregate FVB/NJ, SWR/J, and DBA/2J strains in a hierarchical clustering analysis

Phenotype correlations (Table 3) suggested that grooming and contextual fear learning phenotypes covaried in our data set. Literature searches revealed Dab1, a gene that contributes to Reelin signaling (Howell et al., 1999), as a likely candidate in determining mouse grooming and contextual fear learning phenotypes because it was identified as a positional candidate for grooming behavior in a QTL mapping study (Delprato et al., 2017) and Dab1 mutant mice exhibit abnormal grooming behavior (Strazielle et al., 2012) and impaired freezing in a contextual fear learning test (Trotter et al., 2013). A hierarchical clustering analysis using publicly available Dab1 SNP data grouped these eight strains by dissimilarity of Dab1 SNPs. Greater distance from a shared node, or branch point, represents greater dissimilarity between strains. FVB/NJ, SWR/J, and DBA/2J strains clustered separately from other strains, suggesting dissimilarity between their Dab1 SNPs and those of other strains (Fig 4).

Figure 4.

Figure 4.

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Hierarchical clustering of strains by Dab1 SNPs obtained from Mouse Phenome Database.

4. DISCUSSION

Rodent strain comparisons are useful tools for the identification of genetic factors influencing phenotypes. However, it is possible that behavioral differences among strains compete with the expression of traditional learning measures, such as freezing during fear conditioning. We quantified freezing, rearing, and grooming immediately prior to fear conditioning training (Baseline period) and during fear conditioning testing (Context, PreCS, and CS periods) and found that genetic background influenced behavioral responses during fear conditioning. Correlations and difference scores comparing behaviors across stages of fear conditioning further demonstrated strain-specific conditioned responses. Strain means for freezing negatively correlated with strain means for rearing and grooming, which could suggest shared genetic substrates underlying behaviors or be a result of behaviors being mutually exclusive. Using publicly available phenotype data, we identified correlations between grooming and learning behaviors, suggesting that these phenotypes covaried within our strain panel. Literature searches revealed Dab1 as a likely candidate gene impacting these phenotypes. To test the influence of Dab1 sequence variation on grooming and learning in our strain panel, we conducted a hierarchical clustering analysis using publicly available Dab1 SNP data. Strains with high grooming during the Context test (FVB/NJ, SWR/J, and DBA/2J) clustered far from other strains. This analysis confirmed that Dab1 sequence variation covaried with grooming phenotypes. Collectively, these findings suggest that genetic background determines various behavioral responses during fear conditioning. These behaviors may be influenced by common genetic factors, such as Dab1, which is likely to be involved in grooming and learning behaviors.

Freezing behavior, a classical measure of learning, was influenced by genetic background across Baseline, Context, PreCS, and CS fear conditioning periods (Fig 1A). Most strains showed significant increases in freezing during the Context and CS periods, which was represented as positive difference scores between observation periods (Fig 1B). Freezing negatively correlated with both rearing and grooming (Tables 1), which could represent shared genetic substrates influencing these behaviors. However, it is important to note that freezing represents the absence of motion, and negative correlations could reflect strain differences in activity. Freezing behavior also correlated across different stages of fear conditioning (Table 2), suggesting that strain differences in freezing were influencing learning-associated freezing responses. Overall, these findings reinforce prior reports that freezing during fear conditioning varies across genetic backgrounds (Bolivar et al., 2001; Nie & Abel, 2001; Owen et al., 1997; Park et al., 2011).

Rearing behavior varied by genetic background across all stages of fear conditioning (Fig 2A). This is consistent with prior studies demonstrating genetic influence on rearing behavior (Delprato et al., 2017). Rearing frequencies were altered by conditioning and stimulus exposure in a strain-dependent manner, which we observed as strain effects on difference scores (Fig 2B). A correlation between Baseline and Context rearing suggested that differences in rearing expressed during the Baseline period influenced rearing during Context testing (Table 2). This finding makes interpretation of Context-specific changes difficult because it is unclear if alterations in Context rearing are the product of a novel learned behavior or a continuation of differences observed during the Baseline period. Nevertheless, across strains rearing usually decreased during Context and Cued periods. One possible explanation for this is that Baseline and PreCS periods represent introductions to novel environments, and it is therefore unsurprising that rearing, an exploratory behavior, is increased in those periods relative to the Context and Cued tests. If this could wholly explain the observed reductions in rearing during Context and Cued tests, then one would expect a gradual reduction in rearing across sequential PreCS and CS periods as opposed to a sudden reduction in rearing in response to the CS. Patterns of rearing across time bins of PreCS and CS periods appear heterogenous, as some strains (such as SM/J) show minor changes in rearing frequency in response to the CS, while other strains (such as SWR/J) show decreases in rearing in response to the CS (Supplemental Fig 3C). The clear response to the CS in some strains could suggest that reduced rearing is an expression of fear in those strains.

Grooming behavior in all stages of fear conditioning was also determined by genetic background (Fig 3A). Prior literature supports strain effects on grooming behavior (Delprato et al., 2017). Unlike rearing difference scores, which were mostly negative, the direction of grooming responses varied across strains (Fig 3B). Within our dataset, only Context and PreCS grooming were correlated, indicating that strain differences in conditioned responses cannot be attributed to Baseline differences in grooming. The lack of significant correlations between Baseline grooming and later grooming responses supports the idea that changes in grooming may be the product of a conditioned response or learning (Table 1). Stressors, depending on the severity, can increase or decrease grooming behavior (Fernández-Teruel & Estanislau, 2016). In high-freezing strains, a decrease in grooming during Context or CS periods is likely a direct consequence of their increase in freezing. In the low-freezing FVB/NJ strain, it is possible that an increase in Context grooming could suggest an expression of fear. It is noteworthy that the increase in grooming observed in FVB/NJ mice is specific to the Context test, and it is not present in the CS test. This could be related to the difference of salience and aversiveness between the Context and CS, or it could be related to the different kinds of learning that they represent. Support for the latter comes from the well-established distinct neurobiological mechanisms that drive Context and CS learning (Mei et al., 2005; Phillips & LeDoux, 1992).

If freezing did not adequately represent fear learning in some strains, then strains that performed poorly in our fear learning task assessed by freezing would likely perform better in other learning assays that use different learning metrics. To determine whether freezing in our study was associated with learning in other studies, we correlated our freezing strain means with publicly available learning data sets on Mouse Phenome Database. We did not find significant associations between our Context or CS freezing data and other learning measures (Table 3; Supplemental Table 1), which suggests that conditioned contextual freezing in our study does not correlate with other learning behaviors across strains. This could occur because the expression of fear learning may include additional behaviors beyond freezing that are not detected when correlating only freezing. This interpretation aligns with prior research, which also reported no correlation between fear conditioning and Morris Water Maze learning behaviors in 20 inbred mouse strains (Owen et al., 1997). Our ability to identify these associations was limited by the paucity of other learning studies using these strains and the limited types of learning examined in publicly available data sets. However, we also did not find clear examples of our low-freezers exhibiting strong learning in other studies. The low-freezing FVB/NJ strain has poor vision (Wong & Brown, 2006) and shows deficits in hippocampal learning tasks using non-freezing measures of learning (Errijgers et al., 2007; Mineur & Crusio, 2002). BTBR, another low-freezing strain, is a mouse model of autism that has attentional impairments, prefrontal cholinergic deficits, reduced brain-derived neurotrophic factor mRNA in the dentate gyrus, and reduced hippocampal cell proliferation (McTighe et al., 2013; Stephenson et al., 2011). Less is known about SWR/J learning abilities, but others have also reported that the SWR/J strain shows impaired freezing after fear conditioning compared to the C57BL/6J strain (Szklarczyk et al., 2015). Correlations between freezing and other publicly available phenotypes also suggested that strain differences in freezing are not likely related to strain differences in anxiety-related and locomotor behaviors (Supplemental Tables 2–3). While it is difficult to form conclusions about the SWR/J strain, these findings collectively suggest that behavioral responses from FVB/NJ and BTBR during fear conditioning may accurately reflect impaired learning capabilities.

It is likely that across the strains examined in this study, shared genetic substrates simultaneously contribute to both learning and grooming or rearing phenotypes. Rearing is an exploratory behavior that is expressed in novel environments (Crusio & van Abeelen, 1986) and can aid in information gathering important for spatial learning (Lever et al., 2006; Renner, 1988). The ability to recognize novel environments is inherently related to contextual memory (Lever et al., 2006), so it may be difficult to separate rearing behavior from contextual fear learning abilities. Both rearing and spatial learning abilities have been associated with hippocampal mossy fiber morphology across mouse strains (Crusio et al., 1989; Delprato et al., 2015). Thus, genetic factors influencing a common substrate, such as hippocampal mossy fibers, are likely to influence both rearing and learning measures. Such shared substrates could contribute to the association of learning and rearing behaviors that were observed in this study, such as the association between Barnes maze learning and rearing in the CS period (Table 3).

Our correlation analyses revealed notable covariation between grooming during fear conditioning and learning phenotypes from other studies (Table 3). Through literature reviews and a clustering analysis, we identified a genetic substrate that may have contributed to both grooming and learning phenotypes. Dab1 (Disabled-1) contributes importantly to Reelin signaling (Howell et al., 1999) and neuronal development (Howell et al., 1997). Separate studies have established its importance in determining both grooming behaviors (Delprato et al., 2017; Strazielle et al., 2012) and contextual fear learning (Trotter et al., 2013). One study examining grooming behavior in 53 BXD recombinant inbred strains (derived from C57BL/6J and DBA/2J parental strains included in our panel) identified Dab1 as a likely candidate for influencing grooming behavior across the strain panel (Delprato et al., 2017). It is noteworthy then that our clustering analysis of a strain panel with greater genetic diversity was able to segregate high-grooming strains from other strains based on Dab1 polymorphisms (Fig 4). FVB/NJ, SWR/J, and DBA/2J had the highest grooming levels during the Context period and were clustered together in our Dab1 SNP analysis. FVB/NJ and SWR/J were low-freezing strains, and while DBA/2J mice were not poor learners in our study, many prior studies have found that they have impaired hippocampal learning abilities (Ammassari-Teule & Caprioli, 1985; Balogh et al., 2002; Upchurch & Wehner, 1988; Wehner et al., 1990). These findings suggest that Dab1 is a strong candidate for a genetic substrate that simultaneously influences grooming and learning behaviors.

The current study did not directly determine whether rearing and grooming behaviors represent conditioned fear responses. Follow-up studies can examine whether rearing and grooming exhibit other signs of conditioned behaviors. For example, a future study could examine whether increased grooming or decreased rearing responses extinguish after re-exposure to conditioned stimuli. Another limitation of this study is that all subjects underwent surgery for implantation and removal of osmotic minipumps, with removals occurring one day prior to fear conditioning training. While only saline was administered, it is possible that this prior experience impacted our results. Additionally, because CS testing always occurred after Context testing, it is possible that behaviors observed in the PreCS and CS periods were affected by prior Context testing. Importantly, only male mice were used in the current study. Because new evidence suggests that male and female rats may exhibit different conditioned fear responses (Gruene et al., 2015), future studies should include female mice to address possible sex-specific behavioral responses in mice. Other behaviors, such as darting, may also be considered. Our informal observations did not detect sufficiently frequent darting behaviors during fear conditioning testing in these male mice to warrant formal quantification. Furthermore, expression of behaviors could vary with different conditioning paradigm parameters such as number of trials, and we only examined one conditioning paradigm in this study. Gruene and colleagues (2015), who examined darting behavior during cued fear conditioning training and testing in rats, found that darting behaviors increased with more CS-US pairings. Perhaps a different conditioning paradigm with more CS-US pairings would have revealed more darting behavior or a different behavioral profile than what we have reported. While future research can expand on these ideas, this study is an important step forward in identifying the genetic underpinnings of behaviors exhibited during fear conditioning.

5. CONCLUSIONS

We found that the expression of freezing, rearing, and grooming behaviors during fear conditioning varied across eight inbred mouse strains. Strain differences in baseline behavior and in conditioned responding influenced these behaviors across conditioning periods. Taken collectively, our findings suggest that behaviors outside of the traditional freezing measure may contribute to conditioned fear expression either by explicitly being a conditioned response or indirectly by competing with expression of conditioned responses, and this is likely dependent on genetic background. We also identified Dab1, a gene involved in Reelin signaling, as a likely genetic factor influencing grooming and learning behaviors in inbred mice. In summary, genetic background determines freezing, rearing, and grooming responses during fear conditioning, and it likely exerts these effects through common genetic factors such as Dab1. These findings emphasize the diversity of behavioral responses during fear conditioning across a genetically diverse population.

Supplementary Material

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HIGHLIGHTS.

  • Freezing, rearing, and grooming frequencies differ across mouse strains.

  • Patterns of behavior expressed across stages of fear conditioning vary by strain.

  • Shared genetic substrates may underlie fear conditioning behaviors.

6.ACKNOWLEDGEMENTS

This paper is in memory of David Bucci, Ph.D., a behavioral neuroscientist dedicated to the importance of analysis of behavior and neural substrates. Your time with us Dave was too short.

This study was supported by the National Institutes of Health [U01DA044399 (G.P. & T.J.G.), U01DA041632 (T.J.G) and T32GM108563 (L.R.S.)], the Jean Phillips Shibley Endowment (T.J.G.), and Penn State University. We thank Dr. Dana Zeid, Dr. Lisa Goldberg, and Dr. Sheree Logue for their careful review of this manuscript.

Footnotes

CRediT Statement

Laurel R. Seemiller: Conceptualization, Methodology, Formal analysis, Investigation, Writing – Original Draft, Writing – Review & Editing, Visualization, Project administration

Sean M. Mooney-Leber: Conceptualization, Methodology, Formal analysis, Investigation, Writing – Review & Editing, Visualization, Project administration

Emily Henry: Conceptualization, Methodology, Formal analysis, Investigation

Anne McGarvey: Conceptualization, Methodology, Formal analysis, Investigation

Abigail Druffner: Formal analysis, Investigation

Gary Peltz: Conceptualization, Resources, Writing – Review & Editing, Funding acquisition

Thomas J. Gould: Conceptualization, Methodology, Resources, Writing – Review & Editing, Supervision, Project administration, Funding acquisition

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