Conditional and unconditional components of aversively motivated freezing, flight and darting in mice

Abstract

Fear conditioning is one of the most frequently used laboratory procedures for modeling learning and memory generally, and anxiety disorders in particular. The conditional response (CR) used in the majority of fear conditioning studies in rodents is freezing. Recently, it has been reported that under certain conditions, running, jumping, or darting replaces freezing as the dominant CR. These findings raise both a critical methodological problem and an important theoretical issue. If only freezing is measured but rodents express their learning with a different response, then significant instances of learning, memory, or fear may be missed. In terms of theory, whatever conditions lead to these different behaviors may be a key to how animals transition between different defensive responses and different emotional states. In mice, we replicated these past results but along with several novel control conditions. Contrary to the prior conclusions, running and darting were primarily a result of nonassociative processes and were actually suppressed by associative learning. Darting and flight were taken to be analogous to nonassociative startle or alpha responses that are potentiated by fear. Additionally, associative processes had some impact on the topography of flight behavior. On the other hand, freezing was the purest reflection of associative learning. We also uncovered a rule that describes when these movements replace freezing: when afraid, freeze until there is a sudden novel change in stimulation, then burst into vigorous flight attempts. This rule may also govern the change from fear to panic.

Keywords: Predatory Imminence; darting; defensive behavior; fear conditioning; learning; memory; mouse; neuroscience.

Figure 1.. Behavioral design and schematic representation of the serial compound conditional stimulus (SCS) used for the Replication Group in Experiment 1.

During training, animals were given 2 days each of five SCS-US pairings. The SCS consisted of a 10 s pure tone (7.5 kHz) followed by a 10 s white noise (75 dB). Immediately upon termination of the white noise-SCS, a footshock US (1 s, 0.9 mA) was delivered. On day 3, the animals were tested with 16 presentations of the SCS without delivering any shocks.

Figure 1—figure supplement 1.. Example traces of velocity (cm/s) measurements obtained via EthoVision across five trials on the first day of training for a mouse in the Replication Group of Experiment 1.

Vertical dotted lines denote stimulus onset times and the horizontal dotted line is the threshold for scoring behavior as a dart (22.9 cm/s). Darting episodes are marked with an *. See Figure 1—figure supplement 1—source data 1.

Figure 2.. Trial-by-trial mean (±SEM) percent freezing, peak activity ratio, and darts per minute throughout all stimulus presentations during training (left panels) and testing (right panels) for the Replication Group (n=7; left half of figure) and the Stimulus Change Group (n=7; right half of figure) in Experiment 1.

See Figure 2—source data 1 & Figure 2—source data 2.

Figure 2—figure supplement 1.. Mean (±SEM) percent freezing, peak activity ratio, and darts per minute throughout training (left panels) and testing (right panels) for the CS Duration Group (n=8) of Experiment 1.

Responding during the 10 s preCS period is represented with open circles, responding during the noise is represented with filled in black circles. See Figure 2—figure supplement 1—source data 1.

Figure 3.. Averaged traces of velocity (cm/s) across the first four trials of extinction during testing for Experiment 1.

Data were averaged across all animals per group and binned into ~0.5 s bins (0.533 s) and presented as mean ± SE. These within-subject error bars are corrected for between-subject variability using methods as described in Morey, 2008. During this test, the Replication Group (n=7) and the Stimulus Change Group (n=7) received the serial conditional stimulus in which a 10 s tone was followed by a 10 s noise. The CS Duration Group (n=8) was only tested with a 10 s noise. See Figure 3—source data 1.

Figure 4.. Mean (± SEM) percent freezing, peak activity ratio, and darting for the test session for Experiment 2 (n=8 per group).

Values are averaged across the 16 trials of extinction during test. p-values and significance were determined through one-way ANOVA. *p<0.05, **p<0.01, ****p<0.0001. See Figure 4—source data 1 & Figure 4—source data 2.

Figure 4—figure supplement 1.. Trial-by-trial mean (± SEM) percent freezing, peak activity ratio, and darts per minute throughout 16 trials of testing for Experiment 2 (n=8 per group).

See Figure 4—figure supplement 1—source data 1.

Figure 5.. Averaged traces of velocity (cm/s) across the first four trials of extinction during testing for Experiment 2 (n=8 per group).

Data were averaged across all animals per group and binned into ~0.5 s bins (0.533 s) and presented as mean ± SE. These within-subject error bars are corrected for between-subject variability using methods as described in Morey, 2008. During this test, the no shock-noise test, shock only-noise test, and noise-shock noise test groups were tested with a 10 s noise. The shock only-tone test group was tested with a 10 s tone. See Figure 5—source data 1.

Figure 6.. Trial-by-trial mean (± SEM) percent freezing, peak activity ratio, and darting per minute throughout all stimulus presentations during training (left panels) and testing (right panels) for Experiment 3 (n=8 per group).

See (Figure 6—source data 1 & Figure 6—source data 2).

Figure 7.. Averaged traces of velocity (cm/s) across two trials of extinction during testing for Experiment 3 (n=8 per group).

Datawere averaged across all animals per group and binned into ~0.5 s bins (0.533 s) and presented as Mean ± SE. These within-subject error bars are corrected for between-subject variability using methods as described in Morey, 2008. During this test all groups were tested with a 10 s noise CS. See Figure 7—source data 1.

Figure 8.. Averaged traces of velocity (cm/s) across three trials of extinction during testing for Experiment 4 (n=8 per group).

Datawere averaged across all animals per group and binned into ~0.5 s bins (0.533 s) and presented as mean ± SE. These within-subject error bars are corrected for between-subject variability using methods as described in Morey, 2008. During this test, all groups were tested with a 10 s noise CS. See Figure 8—source data 1.

Figure 8—figure supplement 1.. Trial-by-trial mean (± SEM) percent freezing, peak activity ratio, and darting per minute throughout all stimulus presentations during habituation (left panels), training (middle panels), and testing (right panels) for Experiment 4 (n=8 per group).

For habituation, presented are the values during CS presentation for habituation groups and values during the same 10 s period for context exposure groups. For training, presented are the values during CS exposure for the paired groups and values during the same 10 s period for shock only groups. For testing, all presented values are during the 10 s CS presentation. See Figure 8—figure supplement 1—source data 1 and Figure 8—figure supplement 1—source data 2.

Figure 9.. Analysis of dart timing and magnitude.

(A) and (B) represent the magnitude of darts to the tone (n=48 darts) and noise (n=360 darts) stimuli during testing, as well as the reaction to the first shock (n=102 shocks) on day 1 of training. Data are presented as mean ± SE and come from all groups (total n=102 animals) that received shock during training, collapsed across all experiments. p-values and significance were determined through Welch’s ANOVA. *p<0.05, ****p<0.0001. (C) represents the magnitude (mean ± SE) of the first and second dart within a single CS presentation for all animals across all experiments that performed two darts within a single 10 s CS period (n=65 “multi-darts”). p-values and significance were determined through a paired sample t-test. *p<0.05. (D) and (E) represent the magnitude of darts that occurred during the initial 3 s of the 10 s CS period (n=230 darts) and those that occurred during the final 7 s of the 10 s CS period (n=178 darts). Data are presented as mean ± SE and come from all groups that received shock during training (n=102 animals), displayed by group and stimulus type in (D) and collapsed across all experimental groups in (E). p-values and significance were determined through Welch’s ANOVA. ****p<0.0001. See Figure 9—source data 1.

Figure 10.. Mean (± SEM) percent freezing during extinction/testing for Experiment 2 shows that the occurrence of the stimuli at test disrupt freezing to the context and that the noise disrupts freezing to a greater extent than the tone (n=8 per group).

Also plotted is a similar curve showing freezing and the impact that shock presentation during training has on freezing. These data are averaged across both shock only groups (total n=16) on day 2 trials after fear to the context had been established, showing that shock disrupts freezing to an even greater extent than the noise. See Figure 10—source data 1.

Figure 11.. Trial-by-trial mean (± SEM), peak activity ratio (PAR), and darting per minute throughout all stimulus presentations during training (left panels), and testing (middle panels) for all groups across experiments that received noise-shock pairings, grouped by sex of the animal (n=15 females; n=16 males).

The right panels show individual values for each animal’s average PAR and darts per minute across training and testing. p-values and significance were determined through repeated measures ANOVA, and the interaction was followed up with pairwise t-tests. *p<0.05. See Figure 11—source data 1.