1.9 Passive avoidance learning
Step-through passive avoidance learning is another type o f fear learning that depends on amygdala function (Jellestad and Bakke, 1985). For training in this task, the mouse is placed in the lit compartment o f a passive avoidance system (also see chapter 2). The lit compartment is connected to a dark compartment via a sliding door. Rodents prefer to be in the dark and the animal therefore rapidly enters the dark compartment o f the passive avoidance system. Upon entry the sliding door is closed and the animal receives a mild foot shock. I f placed back into the lit compartment after training, the mouse will avoid entering the dark compartment. The task has been termed passive avoidance, because the animal can remain passive to avoid being shocked. In the active avoidance task, the mouse would be placed into the dark compartment and receive a foot shock. Upon replacement into the dark it would avoid the shock by actively escaping into the lit com partm ent. Sim ilar to contextual fear conditioning, robust learning can be triggered with a single trial and the task can therefore be used to dissociate mechanisms o f STM from those important to LTM formation.
Lesions o f the fornix in rats lead to a selective LTM impairment in this task, suggesting that disconnecting the hippocampal formation impaires passive avoidance memory. The memory impairment coincides w ith impaired activation o f CREB in hippocampus (Taubenfeld et al., 1999). Training in passive avoidance leads to transcription o f C/EBPp in hippocam pal form ation, an immediate early gene regulated by CREB (Taubenfeld et al., 2001). Furthermore, CRE m ediated transcription is activated by passive avoidance learning in both hippocampus and in the amygdala. In contrast to fear conditioning, how ever, passive avoidance learning stim ulates CRE m ediated transcription in dentate gyrus, suggesting that the m olecular mechanisms o f LTM formation differ between the two tasks (Impey et al., 1998).
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1.10 Effects of genetic background on learning and memory
M ost commonly inbred mouse strains are used in the laboratory. These strains have been inbred for specific traits and may have accum ulated mutations that impair performance in certain L&M tasks. For example, most 129 and DBA strains show poor spatial learning (Upchurch and Wehner, 1988; Wolfer et al., 1997; Owen et al., 1997; Nguyen et al., 2000). C57BL/6J and 129/SvEms are good spatial learners and they show robust memory for cued and contextual fear conditioning (Nguyen et al., 2000), although C57BL/6J mice may tend to become deaf to certain frequencies at an early age (Willott, 1986).
The im pact o f a m utation on behaviour in m ice can be influenced by genetic
background. This confound became particularly apparent in the analysis o f L&M in
CREB a/A hypomorphic mice. These mice were initially studied in a mixed C57BL/6 x 129/SvEv background and found to be viable with no obvious abnormalities. They were found to display a selective deficit in LTM at 24 h, but not at 30 min (for contextual conditioning) or Ih (for cued fear conditioning) after training. Furthermore, the mice were impaired in spatial learning in the Morris water maze (Bourtchuladze et al., 1994). This result could be confirmed in a predominantly C57BL/6 background (although the CREBa/A mutation is lethal in a pure C57BL/6 background) and in an F2 background o f C57BL/6 and 129/SvJ, respectively (Kogan et al., 1997). However, subsequent analysis in the C57BL/6 x FVB/N FI background revealed no obvious impairment in fear conditioning and spatial learning in CREB a/A hypomorphic mice, although further reduction o f total CREB levels led to a selective LTM deficit (Gass et al., 1998). A recent study in another C57BL/6 x 129 FI background did find impairments as early as 5 min after training (Graves et al., 2002). Surprisingly, CREBa/A hypomorphic mice in this background had normal spatial learning in the M orris water maze. This indicates that the observed phenotype critically depends on the genetic background o f the animals. Deletion o f the a - and A-isoforms o f CREB in mice led to an up-regulation of
Chapter 1 : introduction to mouse behavioural analysis 53
CREEP and o f the cAMP responsive element modulator CREM (Hummler et al., 1994; Blendy et al., 1996). A possible explanation for the different phenotypes observed in CREBa/A hypom orphic mice could be, that the phenotypes o f the mice may be influenced by compensatory mechanisms supported by different genetic backgrounds.
H ybrid strains can be used to initially characterize the impact o f a mutation on L&M.
Hybrid strains eliminate homozygosity o f recessive mutations that may be deleterious to learning and memory in inbred strains (Silva et al., 1997). In m ost gene-targeting experim ents the m utation is introduced in ES cells derived from mice in a 129 background. Chimeric mice generated from these ES cells can be bred into the C57BL/6 background and the characterisation o f the m utation can be achieved in the F2 generation. However, one drawback o f this strategy is that the mutant locus including neighbouring genes will be derived from the ES cells in a 129 background, whereas the corresponding WT locus will be o f C57BL/6 origin (fig. 1-4). Polymorphisms o f neighbouring genes may influence the behavioural phenotype o f mutant mice and lead to m isinterpretations concerning the function o f a gene for L&M (Gerlai, 1996). A possible solution to the problem has been suggested at the “Banbury Conference on Genetic Background in M ice” held in 1997 at Cold Spring Harbour, New York. Accordingly, the best controls for the F2 homozygotes are WT mice that also have the genomic region linked to the mutated locus derived from 129 background (Silva et al.,
1997). If the phenotypes o f the WT littermate controls and the controls harbouring the 129 derived WT locus do not differ, future experiments can simply use WT littermates as controls. The Cre/lox technology allows for the generation o f null mutant mice and mice having the gene o f interest flanked by loxP sites (floxed) in one targeting step (see introduction part C). The homozygous floxed mice will have the same flanking genes as the null mutants but expression o f the mutated gene will be preserved. Hence, floxed mice can serve as a perfect control for neighbouring gene effects. Alternatively, breeding schemes have been developed to control for the effect o f flanking genes.