Materials and Methods

Mouse lines
The R26R line [85] was employed as Cre indicator. GluR-B ΔECS:FB mice were of mixed C57Bl/6 and NMRI genetic background and generated from TgCre4 (“Camkcre4” in [59]) and GluR-B+/neo [60] mice. GluR-B+/neo mice carry a wild-type GluR-B allele and a gene-targeted GluR-B allele in which the intron 11 sequence critical for Q/R site editing is replaced by a TK-neo gene flanked by loxP sites (“floxed”). GluR-B2lox mice [86] carry gene-targeted GluR-B alleles in which exon 11 is floxed. GluR-B ΔFB mice were of C57Bl/6 genetic background and generated from TgCre4 and GluR-B2lox mice. TgCN12-itTA mice were generated as described for TgCN10-itTA mice (see Materials and Methods in [58]), and represent another founder line obtained from the same pronucleus injection, with more widespread forebrain expression. For exogeneous expression of GFPGluR-B, the mouse line TgOCN1 was generated: an AseI fragment of plasmid pnlacZ/GFPGluR-B was injected into the pronucleus of oocytes obtained from DBA1/C57Bl/6 F1 hybrids. Positive founders were backcrossed into C57Bl/6 for further analysis. Plasmid pnlacZ/GFPGluR-B was constructed from pnlacZ/GFPGluR-A [87] by replacing GluR-A cDNA with the rat cDNA for GluR-B. Transgenic GFPGluR-B protein levels were measured in hippocampus of TgOCN1 mice also carrying a transgene for forebrain-specific homogeneous tTA expression [88].

Experimental groups
Mice heterozygous for TgCre4 and heterozygous for the TK-neo cassette in the GluR-B allele (GluR-BΔECS:FB) or homozygous for the floxed GluR-B (GluR-BΔFB) were used in the experiments. The “rescue” mice (GluR-BRescue) were positive for TgCre4, homozygous for the floxed GluR-B gene, and positive for the tet-sensitive responder transgene TgOCN1 and for the itTA expressing activator transgene TgCN12-itTA.

Control groups
Littermate controls were used in all experiments. GluR-B+/+ mice were used as controls in the task described in Figure 1. GluR-B+/2lox and GluR-B2lox/2lox (both negative for TgCre4) mice were used as controls in experiments described in Figures 2, 4, and 6. For the GluR-BRescue experiments (Figure 6), controls positive for either TgCN12-itTA or TgOCN1 in a GluR-B+/2lox and GluR-B2lox/2lox (both negative for TgCre4) background were used.

Genotyping
Mice were selected by PCR of mouse-tail DNA with specific primers as described below. Indicated are the sequences and the approximate length of the amplified DNA fragments. TgCre4: rspCre1 (5′- ACCAGGTTCGTTCACTCATGG-3′) and rspCre2 (5′- AGGCTAAGTGCCTTCTCTACAC-3′), 200 basepairs (bp).
GluR-Bneo: MH60 (5′- CACTCACAGCAATGAAGCAGGAC-3′), MH53a (5′- GAATGTTGATCATGTGTTTCCCTG-3′), and MH117 (5′- GTTCGAATTCGCCAATGACAAGACG-3′), wild-type: 500 bp and mutant: 400 bp.
GluR-B2lox: VM12 (5′- GCGTAAGCCTGTGA AATACCTG-3′) and VM10 (5′- GTTGTCTAACAAGTTGTTGACC-3′), wild-type: 250 bp and mutant: 350 bp.
TgOCN1: VM4 (5′- CTCCCAGACAACCATTACCTGTCC-3′) and GluR-B882BST (5′- CGAAGTATACTTAATTGTCGCTGTGTG-3′), 600 bp.
TgCN12-itTA: htTA1 (5′- AGAGCAAAGTCATCAACTCTG-3′) and htTA2 (5′- GTGAGAGCCAGACTCACATTTCA-3′), 1,000 bp.

Southern blot analysis
Genomic DNA from mouse-tail/liver was digested with restriction enzyme BglII (NEB), and the Southern blot was done with a 320-bp probe (“integ”) obtained by PCR detecting the αCaMKII promoter.

Histochemistry
Histochemistry was performed as described previously [89], with the following exceptions: Coronal 70- to 100-μm vibratome slices were used for immunohistochemistry with GluR-B (1:60, polyclonal; Chemicon, Temecula, California, United States), GFP (1:8,000, polyclonal; MobiTech, Göttingen, Germany), and Cre (1:8,000, polyclonal; BAbCO, Berkeley, California, United States) primary antibodies, and FITC-coupled (1:200; Dianova, Hamburg, Germany) and peroxidase-coupled (1:600; Vector, Burlingame, California, United States) secondary goat anti-rabbit antibodies.
The main olfactory epithelium was obtained via cryostat sectioning, and immunohistochemistry was performed (primary antibody Cre, 1:5,000, polyclonal; BAbCO). X-gal staining was performed as described [89].

Immunoblot analysis
Mouse brains were removed, and the hippocampus, olfactory bulb, and remaining forebrain areas were isolated. Total protein was prepared, and immunoblots were performed as described [87]. Antibodies used were against GluR-B (1:800, monoclonal; Chemicon), β-actin (1:40,000, monoclonal; Sigma, St. Louis, Missouri, United States) as an internal standard, and secondary goat anti-rabbit and goat anti-mouse antibodies (Vector, 1:15,000). Immunoreactivity was detected with ECLplus (Amersham, Little Chalfont, United Kingdom), and immunoblots were scanned and quantitatively analyzed with ImageJ.

Behavioral analysis: Subjects
All mice were four to six weeks old at the beginning of the experiments. Subjects were maintained on a 12-h light-dark cycle in isolated cages in a temperature and humidity-controlled animal facility. All behavioral training was conducted during daytime. During the training period, animals were kept on free food but on a water-restriction schedule designed to keep them at > 85% of their free food body weight. Continuous water restriction was never longer than 12 h. All animal care and procedures were in accordance with the animal ethics guidelines of the Max-Planck Society.

Apparatus
All olfactory discrimination experiments were performed using three modified eight- channel olfactometers ([61], Knosys, Bethesda, Maryland, United States of America) operated by custom-written software in Igor (Wave Metrics, Lake Oswego, Oregon, United States of America) on Pentium I, II, and III PCs running Microsoft Windows 98. Great care was taken to counterbalance groups between setups. In brief, animals were presented with odor from one out of eight possible odor channels and rewarded with a 2- to 4-μl drop of water in a combined odor/reward port (Figure 1B), ensuring tight association of the water-reward with a presented odorant. Head insertion into the port was monitored by an IR beam and photodiode (Figure 1B). Odors used were n-amyl acetate, ethyl butyrate, pelargonic acid, valeric acid, and binary mixtures of cineol and eugenol. If not otherwise noted, odors were diluted to 1% in mineral oil (Fluka Chemie, Steinheim, Germany) and further diluted by airflow to a final concentration of approximately 0.15%. All dilutions in the text refer to the dilution in mineral oil. All chemicals were obtained from Fluka Chemie.

Task habituation training
Beginning 1–3 d after the start of the water restriction schedule, animals were trained using standard operant-conditioning procedures [3]. In a first pretraining step, each lick at the water delivery tube was rewarded. After 20 licks, a second stage was entered in which head insertion initiated a 2-s “odor” presentation during which a lick was rewarded. The “odorant” used in the pretraining was the carrier medium mineral oil. All animals learned this task within one day (2–3 sessions 30 min each).

Structure of an individual trial
The mouse initiates each trial by breaking a light barrier at the opening of the sampling port (see also [3]). This opens one of eight odor valves, and a diversion valve that diverts all air flow away from the animal for typically 500 ms. After the release of the diversion valve, the odor is accessible to the animal for 2,000 ms. If it continuously licks at the lick port during this time (once in at least three out of four 500-ms bins), it can receive a 2- to 4-μl water reward after the end of the 2,000-ms period. If the animal does not continuously lick, or if the presented odor was a designated nonrewarded odor, neither a reward is given nor any sort of punishment, to minimize stress for the animal. Trials are counted as correct if the animal licks continuously upon presentation of a rewarded odor or does not lick continuously with a nonrewarded odor. A second trial cannot be initiated unless an intertrial interval of at least 5 s has passed. This interval is sufficiently long so that animals typically retract quickly after the end of the trial. Odors are presented in a pseudo-randomized scheme (no more than two successive presentations of the same odor, equal numbers within each 20-trial block). No intrinsic preference toward any of the odors was observed but controlled for by counterbalancing.
A total of 100–300 trials were performed each day, separated into 30- to 40-min stretches to ensure maximal motivation despite the mild water restriction scheme. Additionally, motivation was controlled by monitoring intertrial intervals and the response frequency [3].

Measurement of performance
The simplest measure of performance is the fraction of trials in which the animal responds correctly—that is, responds with licking to the presentation of the S+ odor and does not lick with presentation of the S− odor.
It was shown previously, however, that the detailed sampling pattern is a more sensitive measure of discrimination performance [3]. To avoid long (> 3-week) training periods, we chose not to measure discrimination times [3] but to analyze the average sampling behavior in total. Upon presentation of a rewarded odor, the animal usually continuously breaks the beam, whereas upon presentation of an unrewarded odor the head is quickly retracted. The difference in response to the rewarded and unrewarded odor is approximately sigmoidal (Figures 1D, 1E, and 2D) and yields a sensitive measure of the discrimination performance. From this difference or from a sigmoidal fit to the difference (Figure 1E), several measures of discrimination can be determined: the average difference, peak, or maximum, time of half maximum, and slope of the fitted sigmoid. Whereas for small trial numbers (< 200) the slope often is not well constrained, any of the other parameters yielded essentially the same results. The discrimination index plotted in Figures 1F, 2C, 4D, and 6C refers to the fitted maximum, generally ranging from zero to one, one indicating the best discrimination. Identical results were obtained with other measures of discrimination, such as the average sampling difference.

Structure of training
After habituation, mice were trained to discriminate 1% amylacetate from 1% ethylbutyrate for 500 trials. During the last 100 trials, the S+ odor was rewarded in only 50% of the cases to increase the resistance to extinction of the acquired memory. These trials were excluded for the statistical analysis of the learning curves. Inclusion did not alter the result of the ANOVA; however, linear fitting of the learning curve was not appropriate anymore as partial saturation of learning performance had already occurred. Subsequently, animals were trained for 500 trials on the “difficult” discrimination task [3] between the binary mixtures 0.6% eugenol/0.4% cineol and 0.4% eugenol/0.6% cineol. To allow comparison, the last 100 trials were altered as for the “simple” discrimination task above. After two days of rest, animals were finally trained on the “simple” discrimination task between 1% pelargonic acid and 1% valeric acid for another 600 trials.
In all experiments, counterbalancing between both odors and setups was ensured within and between genetic groups, or results were compared with counterbalanced subgroups, which in every case yielded identical results. During the entire course of the experiment, the person handling the animals and operating the olfactometers was blind to the genotype of the mice.

Memory measurement
To assess memory, after 280 trials of training to discriminate between pelargonic acid and valeric acid, memory trials were interleaved for 120 trials; that is, within each block of 20 trials two unrewarded amylacetate and two unrewarded ethylbutyrate trials were included. Memory scores are given as the fraction of those unrewarded trials that were responded to “correctly” (licking response to the odor that was rewarded in the initial training session [S+], no response to the odor that was not rewarded initially [S−]). Due to the epileptic phenotype and the slightly increased mortality [58], GluRBΔECS:FB mice were trained only for the initial period of 400 trials.

Statistics
Learning curves for both correct performance (“percentage correct”) and the discrimination performance were analyzed by repeated measure ANOVA. Additionally, learning curves were assessed by linearly fitting of trend lines to the data with fixed offsets, leaving the slope as the only variable. In general, binning was 100 trials per block. To allow for the investigation of group/block interactions, the repeated measure ANOVA binning was reduced to 20 trials per block. To compare memory performance in the GluR-BRescue (n = 8) and GluR-BΔFB (n = 22) mice, due to the high variability, a bootstrap approach was employed. Subpopulations of eight animals were selected from the population of 22 GluR-BΔFB mice, and mean memory was determined. In only 343 out of 20,000 subpopulations, mean memory exceeded the mean GluR-BRescue memory of 74.99%, resulting in a p value of p = 343/20,000 = 0.017.