Research report
Generalisation of conditioned fear and its behavioural expression in mice

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Abstract

Mice are favourite subjects in molecular and genetic memory research and frequently studied with classical fear conditioning paradigms that use an auditory cue (conditioned stimulus, CS+) to predict an aversive, unconditioned stimulus (US). Yet the conditions that control fear memory specificity and generalisation and their behavioural expression in such conditioned mice have not been analysed systematically. In the current study we addressed these issues in the most widely used mouse strain of behavioural genetics, C57Bl/6. In keeping with findings in other species we demonstrate the dependence of fear memory generalisation on training intensity (i.e. both US intensity and the number of CS+ and US applied) after both excitatory (explicitly paired presentation of CS+ and US) and inhibitory training (explicitly unpaired presentation of CS+ and US). Furthermore, inhibitory overtraining was associated with changes of uncued anxiety-like behaviour in a light/dark exploration test, indicative of an emotional sensitisation reaction as consequence of a lack of US predictability. Together our results describe the qualitatively and quantitatively different increases of defensive behaviour in response to conditioned stimuli of different salience and identify training conditions that lead to fear memory generalisation and emotional sensitisation in C57Bl/6 inbred mice.

Introduction

Classical (“Pavlovian”) fear conditioning is a firmly established learning paradigm in which subjects quickly learn to associate a previously neutral sensory stimulus (the CS+) or context with a coinciding aversive stimulus (the US). In conditioned subjects subsequent presentations of the CS+ alone according to its predictive value for the US will suppress ongoing, for example, exploratory, behaviours and evoke defensive responses accompanied by autonomic and endocrine arousal (reviewed in [28]). Classical fear conditioning can be applied to various species and has been widely used to investigate how information about aversive emotional experiences is processed and stored in the brain. It has been configured as a model system in many laboratories to study cellular mechanisms of memory formation and to emulate characteristics of psychiatric diseases, such as post-traumatic stress disorder [11], [20]. Over the last years, careful anatomical and physiological studies have identified the neural circuitry that underlies classical fear conditioning [28] and neurochemical and cellular processes in the amygdala that appear to be critically involved (summarised in [15], [35]). The analysis of fear conditioning-induced gene-expression and mutant mouse behaviour have moreover identified some of the molecular factors that play a role in these learning processes [39].

Gene-targeting and transgenic approaches in particular have been fruitful for elucidating cellular and molecular mechanisms of fear conditioning [24] and their relevance for anxiety disorders [13]; however, the induction and expression of fear responses in mice, and the conditions that control stimulus specificity and generalisation have not been analysed as systematically as in other species. Understanding these processes, on the other hand, may be critical for differentiating between processes that specifically relate to associative fear learning or alteration of emotional state and therefore may bear considerable clinical relevance [20]. Previous studies have begun to address these issues by analysing strain differences in fear conditioning to tones and context [19], [29], [37] or generalisation of contextual fear memory [31] in inbred mice.

The objective of the current study was to extend these investigations onto effects of fear conditioning intensity [16] on (1) the intra-dimensional generalisation of auditory cued fear memory and (2) a possible emotional sensitisation and development of generalised anxiety. As experimental subjects we chose inbred mice from the most widely used strain in behavioural genetics, C57Bl/6. Animals were familiarised with the training context and the non-reinforced auditory test stimulus (CS), using a pre-exposure protocol that did not affect their learning performance. They then underwent non-discriminatory conditioning, varied systematically in its intensity with different numbers of explicit CS+/US pairings and US intensities, and were subsequently tested for generalisation to training context and CS. Changes in anxiety-like behaviour were determined after the end of the fear conditioning experiment in a light/dark exploration test.

Section snippets

Material and methods

Adult male C57B/6J OlaHsd mice were used in all experiments. Animals were obtained at an age of 7 weeks (Harlan Winkelmann, Borchen, Germany) and kept in our animal facility for 10 days before the experiment. Mice were housed individually under a 12 h light/12 h dark cycle with lights on at 7:00 p.m. and food and water provided ad libitum. Experiments were always performed between 10:00 a.m. and 6:00 p.m., that is, during the dark cycle. All experiments were in accordance with NIH guidelines and

Behavioural parameters of conditioned fear

As a critical first step in the analysis, we identified behavioural parameters that can be used to describe changes in the defensive behaviour of C57B/6 mice. Various defensive (risk assessment, freezing, flight) and exploratory behaviours, as well as motor activity measures [4], [19] were recorded.

The evaluation during the pre-conditioning period revealed that animals from all experimental groups displayed little or no-fear-related behaviour but spent most of their time with exploratory

Discussion

We examined the generalisation of auditory cued fear memory in the most popular mouse strain of behavioural genetics, C57Bl/6. Our results describe qualitatively and quantitatively different defensive behaviour of conditioned C57B/6 mice to test stimuli of different salience and identify stimulus conditions that result in fear memory generalisation and emotional sensitisation in these animals.

Conclusions

In summary, we have demonstrated three aspects of fear memory after overtraining of C57Bl/6 mice in a classical fear conditioning paradigm: (1) an enhanced defensive response after excitatory conditioning, expressed in flight responses to the conditioned stimulus and in intra-dimensional generalisation to familiar cues and (2) a deficit in contextual and in cued inhibitory conditioning after explicitly unpaired overtraining that was (3) associated with changes of anxiety-like behaviour

Acknowledgements

We thank Dr. D. Wolfer for providing a time-line version of the Wintrack program, Dr. H. Welzl for helpful discussions and comments on the manuscript, and E. Friedel for excellent animal care. This work was supported by a grant from the Leibniz Programm of the Deutsche Forschungsgemeinschaft (to HCP).

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    The authors contributed equally to the paper.

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