Odor Learning and Discrimination Is Enhanced in <i>GluR-B^ΔECS:FB</i> Mice

<div><p>(A) Schematic diagram depicting Cre-mediated activation of GluR-B(Q) by removing the loxP-flanked TK-neo (TK-neo pA, GluR-B<i>^neo</i>) element in intron 11, which is acting as a suppressor in expression from the Q/R site editing deficient <i>GluR-B^neo</i> allele. Exon 10 and 11 encode membrane-spanning segments 1, 2, and 3 (M1, 2, 3) of the GluR-B subunit. The intronic element necessary for editing the Q/R site is shown for the wild-type allele (+).</p> <p>(B) Scheme of an individual trial. Breaking a light barrier, the mouse initiates a trial. An odor is presented, and (depending on the odor denotation and the mouse's response) the mouse is rewarded or retracts its head. A small (2- to 4-μl) water reward is given at the end of an S+ odor if the mouse continuously licks at the delivery tube during the 2-s trial. A trial is counted as correct if the mouse licks continuously upon presentation of a rewarded (S+) odor or does not lick continuously with a nonrewarded (S−) odor [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-b03" target="_blank">3</a>].</p> <p>(C) Twelve experimentally naïve animals (six <i>GluR-B^ΔECS:FB</i> [orange] and six <i>GluR-B^+/+</i> littermate controls [black]) were trained on 1% AA versus 1% EB for two tasks of 200 trials each. Both groups acquired the task (> 70% correct); however, the <i>GluR-B^ΔECS:FB</i> were both quicker, and performed better overall, than the littermate controls (group effect: F_{(1,10 )} = 10.2; <i>p</i> < 0.01). AA, amylacetate; EB, ethylbutyrate.</p> <p>(D) Average head position for one mouse and 50 presentations of the S+ (green) and 50 presentations of the S− (red) odor. “1” indicates the breaking of the light beam (head in the sampling port [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-b03" target="_blank">3</a>]). Note the rapid head retraction for the S− odor.</p> <p>(E) Difference of the average head positions from (D) for S+ and S− odors. Blue line indicates sigmoidal fit. “Discrimination index” refers to the maximum of the fitted sigmoid.</p> <p>(F) As (C) but depicting the discrimination index as a function of trial number (group effect: F_(1,10) = 1.7; <i>p</i> < 0.01).</p></div

Specific Hippocampus and Piriform Cortex Expression of Transgenic ^GFPGluR-B

<div><p>(A) Schematic diagrams depicting forebrain-specific GluR-B deletion as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g002" target="_blank">Figure 2</a>A and itTA-dependent expression of ^GFPGluR-B and nuclear-localized β-galactosidase (nLacZ) in <i>GluR-B^ΔFB</i> mice (termed <i>GluR-B^Rescue</i>). ^GFPGluR-B and nLacZ are both encoded by <i>Tg^OCN1,</i> and itTA is controlled by a fusion of the NR2C silencer element [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-b91" target="_blank">91</a>] and the αCaMKII promoter (termed <i>Tg^CN12-itTA</i> [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-b92" target="_blank">92</a>]).</p> <p>(B) In coronal brain sections of mice positive for both transgenes <i>(Tg^CN12-itTA</i> and <i>Tg^OCN1)</i> β-galactosidase activity (blue, X-gal, counterstain by eosin) is restricted to hippocampal neurons in CA1, DG, and neurons in the piriform cortex. The same neurons show ^GFPGluR-B expression when analyzed in immunohistochemical sections with an antibody against GFP. Scale bars: 500 μm.</p> <p>(C) Immunoblot detecting endogenous GluR-B and transgenic ^GFPGluR-B in the hippocampus of three different mice (#1, #2, #3).</p> <p>(D) Relative quantification from (C) of transgenic ^GFPGluR-B compared with endogenous GluR-B in the hippocampus.</p></div

Odor Learning and Discrimination Is Enhanced, but Odor Memory Is Reduced in <i>GluR-B^ΔFB</i> Mice

<div><p>(A) Schematic diagram depicting Cre-mediated ablation of loxP-flanked exon 11 of the GluR-B alleles.</p> <p>(B and C) Nine <i>GluR-B^ΔFB</i> (red) and nine <i>GluRB^2lox</i> littermate controls (black) were trained for successive odor discrimination tasks on 1% AA versus 1% EB (400 trials), 0.4% Cin/0.6% Eu versus 0.6% Cin/0.4% Eu (400 trials) and 1% Pel versus 1% Val. <i>GluR-B^ΔFB</i> mice showed increased learning/discrimination compared with controls, both using the performance as measured by percentage of correct trials ([B]; group effect: F_1,16 = 6.55, <i>p</i> < 0.05) or the discrimination index (C), that is the maximal difference of the sampling pattern (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#s4" target="_blank">Materials and Methods</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g001" target="_blank">Figure 1</a>D–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g001" target="_blank">1</a>F; F_1,16 = 29.5, <i>p</i> < 10⁻⁴).</p> <p>(D) Sampling difference for the last 100 trials of the mixture discrimination task (indicated with a black arrow in [C]) for all 18 individual mice. Note that the <i>GluR-B^ΔFB</i> mice show a consistently larger sampling difference.</p> <p>(E) Olfactory memory performance for nine littermate controls (black) and nine <i>GluR-B^ΔFB</i> (red) mice. Olfactory memory was tested at the time indicated by the black bar in (C) by interleaving the Pel and Val trials with unrewarded AA and EB trials. AA, amylacetate; Cin, cineol; EB, ethylbutyrate; Eu, eugenol; Pel, pelargonic acid; Val, valeric acid.</p></div

Variability of Cre Expression of Mouse Line <i>Tg^Cre4</i>

<div><p>(A) Variable Cre expression in forebrains of three different mice positive for <i>Tg^Cre4</i> and <i>R26R</i> Cre indicator (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#sg001" target="_blank">Figure S1</a>) 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.</p> <p>(B) Southern blot analysis of BglII-digested genomic mouse DNA of four <i>Tg^Cre4</i> 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.</p></div

Olfactory Memory but not Odor Learning/Discrimination Is Correlated with Residual GluR-B Levels in Hippocampus and Forebrain of <i>GluR-B^ΔFB</i> Mice

<div><p>(A) The olfactory memory performance for 18 <i>GluR-B^ΔFB</i> (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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g002" target="_blank">Figure 2</a> (open symbols) and an additional experiment with nine <i>GluR-B^ΔFB</i> and two littermate controls (shaded symbols).</p> <p>(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 <i>GluR-B^ΔFB</i> (#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 <i>GluR-B^ΔFB</i> 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).</p> <p>(D) From ten <i>GluR-B^ΔFB</i> 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g002" target="_blank">Figure 2</a>C) were plotted against GluR-B levels. Memory was tightly correlated to GluR-B protein level in hippocampus (R² = 0.72; <i>p</i> < 0.003) and cortical forebrain (R² = 0.62; <i>p</i> < 0.006) and only weakly in the olfactory bulb (R² = 0.48; <i>p</i> = 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).</p></div

GluR-B Expression in Hippocampus and Forebrain Partially Rescues the Memory Deficit of <i>GluR-B^ΔFB</i> Mice

<div><p>(A) Olfactory memory experiments as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g002" target="_blank">Figure 2</a> were performed with <i>GluR-B^Rescue</i>. Individual mice are indicated: control <i>GluR-B^2lox</i> with black circles, <i>GluR-B^ΔFB</i> with red triangles, <i>GluR-B^Rescue</i> with green squares. Data were combined from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g003" target="_blank">Figure 3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g004" target="_blank">4</a> (open symbols) and an additional experiment with four <i>GluR-B^ΔFB</i>, three littermate controls, and eight <i>GluR-B^Rescue</i> (shaded symbols).</p> <p>(B) Cumulative histogram of the memory performance. Memory performance is indicated as a stepped line. The sigmoidal fit is indicated as a continuous line. The predicted rescue on the basis of the extent of transgenic ^GFPGluR-B expression (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g005" target="_blank">Figure 5</a>D) and the correlation between memory and GluR-B levels in piriform cortex (9.1 ± 2.5% increase in memory for 10% increase in protein, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g004" target="_blank">Figure 4</a>D) is shown in blue. Note that the predicted rescue is in perfect numerical correspondence to the memory performance of <i>GluR-B^Rescue</i> mice.</p> <p>(C) The discrimination index was calculated as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g001" target="_blank">Figures 1</a>E, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g002" target="_blank">2</a>C, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g004" target="_blank">4</a>D (last 100 trials of the mixture discrimination task) for <i>GluRB^Rescue</i> (<i>n</i> = 8), <i>GluR-B^ΔFB</i> (<i>n</i> = 22), and control (<i>n</i> = 14) animals.</p></div

Inclusion appearance in various cortical and striatal regions in zQ175 heterozygous mice.

<p>(<b>A</b>) Using an automated microscope, whole mouse brain sections were scanned by high resolution multi-image acquisition. Individual images were assigned to distinct areas within the cortex including the cingulate cortex (ccx) and motor cortex (mcx), or within the striatum, including dorsal (d)/ventral (v) and medial (m)/lateral (l) parts to allow region-specific automated multiparametric analysis. (<b>B</b>) Region-specific analysis in the striatum of nuclear mHTT inclusions in MSNs. Inclusion number was found to be significantly higher in lateral quadrants (ld and lv) than in medial ventral quadrant at 8 and 12 months old zQ175 mice. (<b>C</b>, <b>D</b>) Subregion specific analysis in the cortex showing quantification of the number of nuclear (<b>C</b>) and extranuclear (<b>D</b>) mHTT inclusions in the cingulate and motor cortex over time. A significantly higher number of inclusions were detected in the ccx compared to mcx region in zQ175 heterozygous mice at 12 months of age. Data are displayed as mean +/-SD. Statistical analysis was performed by two-way ANOVA and Sidak’s multiple comparisons’ test. Mean values were calculated for every age and region using an n of 8 animals with 6 sections per animal; *p<0.05; **p<0.01; ***p<0.001.</p

Workflow of high content imaging for <i>ex vivo</i> phenotypic characterization.

<p>Mouse brain sections fluorescently stained for up to three target proteins were aligned and mounted in glass-bottom multi-well plates suitable for high content imaging. Using an automated imaging setup (Opera, PerkinElmer) up to 600 high resolution confocal images per brain section were acquired. Automated multi-parametric analysis was applied to every single image to generate comprehensive quantitative data sets for each section describing numbers, subcellular structures, morphology and intensities for stained subpopulation of cells.</p

Quantification of nuclear and extranuclear EM48-ir inclusions in the striatum and cortex of zQ175 heterozygous mice.

<p>Brain sections of zQ175 heterozygous mice up to 12 months of age were subjected to immunohistochemical staining for DARPP-32 and EM48, followed by the analysis of inclusion number, size and distribution. (<b>A</b>) In the striatum the number of nuclear mHtt inclusions are significantly increase between 3 and 4 month of age and between 6 to 8 months, reaching a plateau at 12 month. (<b>B</b>) Striatal nuclear inclusions increase steadily in size from 4 to 12 months of age with a significant increase occurring between 6 and 8 months. (<b>D</b>) Nuclear mHtt inclusions in the cortex show a delayed kinetics with a significant increase in number observed among 6, 8, and 12 months of age and (<b>E</b>) a significant increase in size from 8 to 12 months of age. The number of extranuclear inclusions was normalized to the total area of cells measured and reported as density values. The density of extranuclear inclusions significantly increased between 4 and 8 months of age in the striatum (<b>C</b>) and between 6 and 8 months of age in the cortex (<b>F</b>). Data are displayed as dot plots with mean +/- SD. Statistical analysis was performed using standard ANOVA and Sidak’s multiple comparisons’ test. For every age an n of 8 animals with 6 sections per animal were used for quantitation; *p<0.05; **p<0.01; ***p<0.001.</p

Image texture parameter “granularity” for analysis of early mHTT aggregation.

<p>(<b>A</b>) Image analysis strategy for early aggregation parameter “granularity”. The area of cell nuclei was detected based on the DAPI signal. The spatial pattern of pixel intensities of nuclear EM48-ir signal was analyzed and a granularity index indicating formation of small EM48-ir inclusion species was calculated using the SER (Spots) texture algorithm (Acapella, PerkinElmer). Brain sections from 2–12 months old zQ175 heterozygote mice were immunolabelled for DARPP-32 and EM48. The “granularity” index in the striatum and cortex was used to detect and monitor early changes in mHTT distribution and signal clustering. (<b>B</b>) A significant increase in the nuclear EM48 “granularity” in the striatum was observed from 2 to 6 months of age followed by a significant decrease from 6 to 12 months. (<b>C</b>) In the cortex, a significant increase in EM48 granularity was observed from 6 to 12 months. Data are displayed as dot plots with mean +/-SD. Statistical analysis performed using standard ANOVA and Sidak’s multiple comparisons’ test. For every age an n of 8 animals with 6 sections per animal were used for quantitation; *p<0.05; **p<0.01; ***p<0.001.</p