Research report
Spatial learning, contextual fear conditioning and conditioned emotional response in Fmr1 knockout mice

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Abstract

Fmr1 knockout mice are an animal model for fragile X syndrome, the most common form of heritable mental retardation in humans. Fmr1 knockout mice exhibit macro-orchidism and cognitive and behavioural deficits reminiscent of the human phenotype. In the present study additional behavioural and cognitive testing was performed. Knockouts and control littermates were subjected to a spatial learning test using a plus-shaped water maze. Animals had to learn the position of a hidden escape platform during training trials. The position of this platform was changed during subsequent reversal trials. Previously reported deficits in reversal learning were replicated, but we also observed significant differences during the acquisition trials. A plus-shaped water maze experiment with daily changing platform positions failed to provide clear evidence for a working memory impairment, putatively underlying the spatial learning deficits. Two different test settings were used to examine the reported deficit of Fmr1 knockout mice in fear conditioning. Conditioned fear responses were observed in a contextual fear test, and the ability to acquire an emotional response was tested by means of response suppression in a conditioned emotional response procedure. Neither protocol revealed significant differences between controls and knockouts.

Introduction

With its incidence of 1 in 4000–6000 fragile X syndrome is the most frequent form of hereditary mental retardation in humans (for review see Ref. [17]). Fragile X syndrome is caused by deletion or transcriptional inactivation of the fragile X mental retardation 1 (FMR1) gene, leading to the absence of FMRP, the protein derived from the FMR1 gene. An animal model with inactivation of the Fmr1 gene was developed by Bakker et al. [1]. Fmr1 knockouts were shown to be negative for Fmr1 RNA in testis, and for FMRP in testis, brain, and other organs tested. They show normal reproductive fitness and do not display major neurological dysfunction. Testicular weight was significantly higher in knockouts than controls, a finding which may relate to the macro-orchidism observed in fragile X men [1].

Fragile X patients exhibit a wide range of clinical characteristics, including moderate to severe mental retardation [7], [8], [13], [17], [19]. Fragile X males and mentally retarded fragile X-negative males have several behavioural and cognitive characteristics in common (including memory deficits), but fragile X patients have relatively better vocabulary and receptive word knowledge and verbal-expressive skills. However, they display inferior visual-motor co-ordination and manual skills, and are less capable of mental reasoning in solving new problems [19]. Since fragile X patients have, indeed, a distinct profile of behavioural and cognitive deficits, several attempts were made to assess these functions in the animal model [1], [3], [6], [9], [16], [23]. Place learning of Fmr1 knockouts and normal littermates was compared in different spatial navigation tasks and training protocols. Fmr1 knockouts showed mildly impaired performance in a Morris-type water maze [1], [6], [16]. Knockouts especially experienced difficulties in learning to locate the hidden platform when, after a period of intensive acquisition training, the platform's position was changed during the reversal trials [1], [6], [16]. However, in a recent publication, Paradee et al. [23] were able to replicate only part of these results, which could have been due to strain differences between C57BL/6 and 129Re/J influencing the Fmr1 knockout phenotype. Also, in a simplified navigation task using an E-shaped water maze, we found no significant differences between Fmr1 knockouts and normal littermates, either during the initial acquisition training, or during reversal training [16]. On the other hand, a preliminary report has mentioned dramatic differences in acquisition performance between knockouts and controls using massed-trial training in a plus-shaped water maze [3]. However, no further confirmation of these observations has ensued, and they might have been due to retinal degeneration in the background strain (T. Brown, personal communication).

Several authors also examined non-spatial learning abilities in Fmr1 knockouts. Passive avoidance learning in the step-through box was shown to be normal [1]. Using operant conditioning techniques in a small number of animals, knockouts were found to be similar or even superior to controls in acquiring visual and auditory discriminative responses [9]. Finally, a recent well-performed study by Paradee et al. [23] did demonstrate deficits in conditioned fear responses in Fmr1 knockouts.

In the present study we have made an effort to replicate previously reported results in Fmr1 knockout mice using different behavioural test protocols. Fmr1 knockouts and normal littermates were tested in two independent plus-shaped water maze experiments using the same initial training protocol as Brown et al. [3]. The initial training was followed by an additional series of reversal trial blocks to examine reversal learning in this task. It was expected that knockouts might show a similar reversal deficit as previously reported using the Morris-type water maze. Secondly, we have examined whether the previously demonstrated reversal deficit could have been due to defective working memory functions, rather than to relative inability of knockouts to change a previously learned navigation strategy [1]. To test this, mice were subjected to a plus-shaped water maze learning protocol with changing platform positions. Finally, the putative deficit of Fmr1 knockouts in fear conditioning was examined. Context-dependent fear conditioning was studied using the same protocol as Paradee et al. [23]. In another series of experiments, the effect was studied of a conditioned fear response component superimposed on a food-reinforced response schedule.

Section snippets

Transgenic animals

Fmr1 knockout mice and wildtype littermates were derived from our previously described line, back-crossed to the C57BL/6JIco inbred strain for at least ten generations [1]. Genotypes were determined by polymerase chain reaction (PCR) and Southern blotting. Male Fmr1 knockout mice and control littermates with an average age of 2–3 months were used. Mixed genotype groups of approximately eight littermates were housed in standard mouse cages under conventional laboratory conditions (food and water

Plus-shaped water maze learning

Both controls and Fmr1 knockout mice were able to learn the location of the platform as a result of training. For the acquisition trials two-way RM-ANOVA revealed a significant effect of trial block on escape latency (F5,65=40.83; P<0.001; Fig. 1A). The effects of genotype and of genotype×trial block on escape latency were not significant (P=0.573, P=0.929, respectively). Two-way RM-ANOVA revealed a significant effect of trial block on the number of correct trials/trial block (F5,65=31.87; P

Discussion

Previous histological and neurocognitive studies identified Fmr1 knockout mice as a putative model for fragile X syndrome, the most common form of inherited mental retardation in man. In the present study we have examined acquisition and reversal learning of plus-shaped water maze navigation in Fmr1 knockouts and their wild-type littermates in order to compare these results with earlier findings in the Morris-type water maze [1], [6], [16]. Plus-shaped water maze training with daily changing

Acknowledgements

This work was supported by the University of Antwerp, Born-Bunge Foundation, the Belgian Fund for Scientific Research-Flanders (FWO Grants G.0027.97 and G.0091-97), the FRAXA Research Foundation, Biomed Grant BMH4-CT96-1663, NeuroSearch Antwerp, and the OCMW-Antwerp Medical Research Foundation. RD is a postdoctoral fellow and DVD a research assistant of the Fund for Scientific Research-Flanders.

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