Olfactory Memory Is Significantly Decreased but Highly Variable in GluR-BΔFB Mice
To capture the full extent of the role of Ca2+-permeable AMPARs in olfactory behavior, we next assessed the effects of altered AMPA channels on olfactory memory. To probe olfactory memory in GluR-BΔFB mice, six days after the end of the first training phase for odor discrimination (amylacetate versus ethylbutyrate), the training trials for the third odor pair (pelargonic versus valeric acid) were interleaved with unrewarded trials in which amylacetate or ethylbutyrate were again presented (black bar in Figure 2C). Whereas control mice reliably responded only to the previously rewarded odor (memory of 86 ± 8%, mean ± SD, n = 9, Figure 2E), GluR-BΔFB mice showed reduced olfactory memory (69 ± 16%, n = 9, p < 0.05, Mann-Whitney). Due to the more rapid learning observed in GluR-BΔFB, one could speculate that a decrease in olfactory memory might simply reflect increased extinction. However, extinction levels were low in general, no significant group-trial interaction could be found for the memory trials (2-way ANOVA, F(6,90) = 1.5, p > 0.1), and a restriction of the analysis to early memory trials displayed essentially the same pattern (Figure S3A). Thus, reduced performance in the probe trials is not due to increased extinction but reflects genuine memory impairment. Moreover, because the hippocampus-dependent spatial memory after-task acquisition in the Y-maze was not affected (Figure S2), we conclude that the observed olfactory memory deficit is rather specific for olfaction and does not readily generalize to other modalities.
While the improved odor discrimination and learning behavior showed only little variability, the significantly impaired memory performance observed in GluR-BΔFB mice was highly variable among individual animals compared with control littermates (Figure 2E). This variability in olfactory memory was reflected in the level and extent of Cre-recombinase expression in forebrain of transgenic TgCre4 mice, as visualized by Cre-activity in the Cre-indicator mouse line R26R. We observed that onset and extent of Cre-recombinase expression in different forebrain regions varied among individual TgCre4 mice (Figure 3A), which could also be directly visualized by immunohistochemistry with a Cre-antibody (unpublished data). As this variability persisted after several backcrosses, and Southern blot analysis revealed no differences of transgene integration or number among animals (Figure 3B), it could not be attributed to genetic differences but rather to epigenetic mechanisms.
Figure 3  Variability of Cre Expression of Mouse Line TgCre4
(A) Variable Cre expression in forebrains of three different mice positive for TgCre4 and R26R Cre indicator (see Figure S1) at postnatal day 12 pictured by the Cre-dependent β-galactosidase activity (blue, X-gal, counterstain by eosin) in coronal brain slices. Scale bar: 1.25 mm.
(B) Southern blot analysis of BglII-digested genomic mouse DNA of four TgCre4 mice that differed in the Cre expression pattern. Southern probe detects the wild-type (4.5 kbp) and the transgenic (7, 5, 3, and 2 kbp) alleles. Hence, we hypothesized that the variability in olfactory memory reflected the mosaicism observed in the transgenic TgCre4 line. The evaluation of regional differences in expression pattern of animals with robust and poor olfactory memory could then be applied to identify brain areas responsible for the observed phenotypes.