Olfactory Memory Correlates with Residual GluR-B Protein Expression in GluR-BΔFB Mice
Thus, to examine whether the pronounced variability of olfactory memory in GluR-BΔFB mice (Figure 2E) reflects variability of GluR-B levels in GluR-BΔFB mice, we analyzed the residual amount of GluR-B protein in mice with disparate memory performances (Figure 4A–C). Notably, mice with pronounced memory deficits (memory < 70%) showed essentially no detectable GluR-B protein in hippocampus, amygdala, olfactory bulb, and piriform cortex (n = 2, Figure 4B, and unpublished data), but mice with almost complete memory displayed substantial residual GluR-B levels in all brain areas investigated (n = 2, Figure 4B, and unpublished data).
Figure 4  Olfactory Memory but not Odor Learning/Discrimination Is Correlated with Residual GluR-B Levels in Hippocampus and Forebrain of GluR-BΔFB Mice
(A) The olfactory memory performance for 18 GluR-BΔFB (red) and 11 littermate control (black) mice is given as mean (thick lines ± SEM) and as individual performance in open circles and triangles. Arrows with numbers (#) indicate those mice used in experiments (B–C). Data were combined from Figure 2 (open symbols) and an additional experiment with nine GluR-BΔFB and two littermate controls (shaded symbols).
(B and C) Residual GluR-B levels as detected by anti-GluR-B immunofluorescence in hippocampus, amygdala, piriform cortex, and olfactory bulb of one control (#1) and two GluR-BΔFB (#2 and #3) coronal mouse brain sections (B) and by immunoblot analysis from hippocampal (Hip), cortical forebrain (FB), and olfactory bulb (OB) protein extracts of control (#4) and GluR-BΔFB mice (#5, #6, #7, and #8) probed with antibodies detecting GluR-B and β-actin as an internal loading control (C). Scale bars: 200 μm (first panel), 100 μm (other panels).
(D) From ten GluR-BΔFB mice, the individual odor learning/discrimination and olfactory memory performance was determined together with the relative GluR-B levels in immunoblots of hippocampal, forebrain, and olfactory bulb protein extracts. Memory performance (top panels) and discrimination capability (bottom panels; discrimination index is measured for the last 100 trials of the mixture discrimination task as indicated by the arrow in Figure 2C) were plotted against GluR-B levels. Memory was tightly correlated to GluR-B protein level in hippocampus (R2 = 0.72; p < 0.003) and cortical forebrain (R2 = 0.62; p < 0.006) and only weakly in the olfactory bulb (R2 = 0.48; p = 0.03). No measure of learning/discrimination (discrimination index for last 100 mixture trials [D], slopes of trend lines, average discrimination index, average sampling pattern differences, correct performance, etc. [not shown]) displayed any correlation (R2 < 0.3). To quantify the relation between residual GluR-B protein and olfactory memory, the memory experiment was repeated with nine additional GluR-BΔFB mice and two GluR-B2lox control animals (indicated with shaded symbols in Figure 4A), resulting in the same mean, variability, and range of memory performance (control: 89 ± 10%; GluR-BΔFB: 63 ± 14%). Subsequently, protein was extracted from olfactory bulbs, cortical areas, and hippocampi from each mouse, and GluR-B protein was quantified (Figure 4C). The summarized correlations are depicted in Figure 4D (two animals were used for immunofluorescent analysis that yielded the same results as in the first experiment). Whereas no learning or discrimination-related parameter correlated with residual protein levels (Figure 4D, R2 < 0.3), a strong correlation between memory and GluR-B protein was observed in hippocampus (Figure 4D, R2 = 0.72, p < 0.003, n = 10) and cortical areas (Figure 4D, R2 = 0.62, p < 0.006, n = 10). Only a weakly significant correlation was found in the olfactory bulb (Figure 4D, R2 = 0.48, p = 0.03, n = 10). GluR-A levels were unchanged from wild-type, indicating that compensatory up-regulation of other AMPAR subunits is unlikely (GluR-A levels relative to control: 1.02 ± 0.05, mean ± SEM, n = 10).
In summary, mice with reduced GluR-B levels in forebrain areas showed decreased olfactory memory, which correlated tightly with a reduction in GluR-B levels in the hippocampus and cortical areas. Enhanced odor learning and discrimination, on the other hand, was independent of residual GluR-B levels in the olfactory bulb and other forebrain areas, indicating that moderate GluR-B reductions are sufficient to saturate enhanced odor learning and discrimination. Thus, although both are mediated by alterations in the AMPAR subunit GluR-B, due to the qualitatively different dose-response curves, the phenotypes regarding olfactory memory, and olfactory learning/discrimination must be mechanistically distinct.