Research report
Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice

https://doi.org/10.1016/S0166-4328(99)00047-9Get rights and content

Abstract

The performance of C57BL/6J (6J), C57BL/6N (6N), DBA/2J (2J) and DBA/2N (2N) mice in context- and tone-dependent fear conditioning was determined 24 h after fear conditioning to evaluate and compare different behavioral measures as indices of emotional learning. Freezing, the change in activity and the size of the explored area were evaluated as behavioral parameters indicating fear. Additionally, the heart rate (HR) increase elicited by tone presentation was evaluated as an autonomic indicator of fear. During the context-dependent memory test, freezing was high only in 6J and 6N mice, whereas a drop of activity and a reduced exploratory area was measured in all strains. During the tone-dependent memory test, high freezing, low activity, reduced exploratory area and a strong HR increase were demonstrated only in 6N and 6J mice, whereas behavioral and HR changes of 2J and 2N mice were always low. In extinction tests, context- and tone-dependent freezing of 6J mice decayed significantly faster than the freezing of 6N mice, whereas in both substrains the conditioned tachycardia to tone extinguished similarly in the home cage. The data demonstrate that monitoring of additional behavioral measures besides freezing and autonomic measures is necessary to interpret differences in associative learning performance of mouse strains that could be related to a differential expression of fear.

Introduction

Investigations of cognitive functions by strategies employing mutant mice require extensive knowledge of the behavior of different strains and substrains [5], [28]. The behavioral performance of mice has been demonstrated to be affected by genetic factors (strain), tasks, and sex [5]. For the investigation of cognitive function, fear-related behaviors have gained increasing interest. Fear conditioning has been demonstrated to be a useful behavioral paradigm to investigate fear-related long-lasting memory function [25]. Fear conditioning is based on the association of a neutral stimulus (conditioned stimulus, CS) such as a defined context (multisensory conditioning) or tone (unisensory conditioning) with an aversive event such as footshock (unconditioned stimulus, US). Fear conditioning is rapidly acquired, it can be performed in different animal species, and the underlying neuronal pathways are assumed to be similar in mammals [13].

All previous comparisons of fear conditioning performance of inbred mouse strains were based on freezing, a defensive behavior of rodents [3], as the only behavioral measure [5], [17], [21], [23]. Genetic differences have been described to affect several defense reactions in different inbred strains including C57BL/6J (6J) and DBA/2J (2J) mice [3], [9]. This may be a consequence of the lack of selection pressure in inbred mice for certain defensive behaviors naturally required to avoid threatening stimuli such as the attraction of predators. Therefore, differences in the defensive behavior repertoire and coping behavior [29] may affect the interpretation of learning and memory performance in comparisons of different mouse strains when limited to the assessment of freezing as the only behavioral parameter. Thus, additional behavioral measures such as activity and area covered during contextual exploration (exploratory area) should provide relevant data based on the well described suppression of exploratory behavior of mice exposed to stressful unconditioned stimuli [12], [16] or after pharmacological induction of stress (anxiety) [6], [10]. Activity measurements were only recently introduced in unsignaled fear conditioning [18], [24], [31], and the explored area has not been evaluated by now as conditioned response in fear conditioning. The stress impact on the endocrine system and autonomic function has been described [6]. However, there were methodological limits in measuring autonomic function, because the experimental procedures and the techniques used were stressful themselves and thereby had a strong influence on the outcome of the experiments as reviewed for rats [11]. However, it was recently demonstrated that a strong heart rate (HR) increase measured in the tone-dependent memory test in the absence of physical activity reflected associative learning in C57BL/6N (6N) mice [30]. In these experiments, HR was monitored telemetrically in the home cage of freely moving mice. This method allowed to eliminate all effects of handling, novelty and contextual cues on HR.

On a genetic basis, different mice may express their fear responses to aversive stimuli in a strain-specific fashion which may vary considerably. For example, fear conditioned DBA/2 and C57BL/6 mice were found to perform differently after re-exposure to context but not tone as conditioned stimuli [23]. Another example is the different generalization of the J and N substrains of C57BL/6 mice [24]. It is unclear whether these differences indicate different learning abilities or coping strategies. It was therefore the objective of this study to extend the experimental comparison of the J and N substrains of DBA/2 and C57BL/6 mice by increasing the repertoire of behavior tests and measures to determine the learning abilities of these mice.

Section snippets

Animals

In this investigation a total of 215 male C57BL/6JOlaHsd (6J; n=59; Harlan-Winkelmann, Borchen, Germany), C57BL/6NCrlBR (6N; n=59; Charles River, Sulzfeld, Germany), DBA/2NCrlBR (2N; n=53; Charles River, Sulzfeld, Germany), and DBA/2JBom (2J; n=44; Bomholtgård, Ry, Denmark) mice were tested. All mice were shipped at an age of 8 weeks and housed individually for 1 week (acclimation) before testing. They were individually housed in standard macrolon cages (type 2: 22×16×13 cm) with free access to

Training (acquisition)

All strains showed a relatively similar behavior during training (Fig. 1). No freezing was scored during the 180-s period in context 1 (Fig. 1A). The low variance of the freezing scores during the 30-s tone presentation did not allow to perform an ANOVA. The mean activity values in context 1 were not significantly different among the four strains, F(3,78)=1.29, P>0.28 (Fig. 1B). However, the exploratory areas covered in context 1 differed significantly among the four strains, F(3,78)=5.93, P

Discussion

The behavioral performance of four mouse substrains derived from two different strains was evaluated and compared in fear conditioning experiments. Besides freezing, additional behavioral and autonomic measures such as activity and HR were included. Activity and HR measurements have been established in the laboratory as indicators of associative learning [18], [24], [30], [31]. Interestingly, 6N mice not conditioned to tone responded to the tone onset with a transient HR increase which was

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