Cryg-immunreactivity in the rat, mouse, and gerbil SOC at P4.

<p>All image series show Cryg-ir (α-Cryg) in the first, VGluT1-ir (α-VGluT1) in the second, and an overlay of both immunoreactivities in the third column. This order applies also to subsequent Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161140#pone.0161140.g003" target="_blank">3</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161140#pone.0161140.g005" target="_blank">5</a>. (<b>A)</b> Cryg-ir is clearly seen in the MNTB and in the ventral acoustic stria of rat. (<b>B)</b> The MSO and a subpopulation of the LSO show also prominent Cryg-ir. (<b>C,D)</b> The mouse displays a weaker labeling in the SOC. (<b>E-F)</b> MNTB, LSO and MSO of the gerbil show no Cryg-ir above background. MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; LSO; lateral superior olive. MNTB, medial nucleus of the trapezoid body, designated by a asterisk MSO, medial superior olive, designated by a star; () LSO; lateral superior olive, designated by a diamond. The abbreviations and symbols also apply to subsequent figures. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.</p

Cryg-ir in the mouse SOC at P25.

<p>All three antibodies against crystallins, i.e. Cryg (<b>A,B</b>), Crygd/e (<b>C,D</b>) or Crygn (<b>E,F</b>) gave no signals above background in the MNTB, the LSO and the MSO of mice aged P25. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.</p

Immunohistochemical and anatomical analysis of the SOC in <i>Crygn</i>^<i>Egr2</i> mice.

<p>(<b>A</b>) Normal gross anatomy of the SOC in <i>Crygn</i>^<i>Egr2</i> mice. GlyT2 and VGluT1 immunoreactivity in coronal brainstem sections of P25 <i>Crygn</i>^<i>Egr2</i> mice and control litter mates indicate normal gross morphology of SOC nuclei. (<b>B</b>) Structural alterations in nuclei of the SOC. In <i>Crygn</i>^<i>Egr2</i> mice, the volumes of the LSO and MNTB were significantly decreased. The MNTB of <i>Crygn</i>^<i>Egr2</i> mice displayed also lower cells number but normal cross sectional area of neurons. At P4, MNTB cell number was not affected by the genotype. Volume and cell number were analysed in Nissl-stained serial sections of the respective nucleus (6 nuclei from 3 animals/genotype). As a test for statistical significance, a two-tailed student’s <i>t</i>-test was used. Color coding (black: control control litter mice; red: <i>Crygn</i>^<i>Egr2</i> mice). Dorsal is up and medial to the left. * <i>p</i> ≤ 0.01; ***<i>p</i> ≤ 0.001.</p

Crygn-ir in the rat, mouse, and gerbil SOC at P4.

<p>Crygn clearly labels the MNTB (<b>A</b>), the LSO and the MSO (<b>B</b>) of rat. (<b>C)</b> The mouse MNTB shows a moderate labeling and the LSO and MSO a weak immunoreactivity (<b>D</b>). (<b>E)</b> Gerbils show a similar pattern of Crygn-ir as the mouse with the MNTB being strongest labeled. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.</p

Altered auditory brainstem responses (ABR) in <i>Crygn</i>^<i>Egr2</i> mice.

<p>(<b>A</b>) Auditory brainstem response (ABR) thresholds for click, noise burst, and pure tone stimuli for control (white) and <i>Crygn</i>^<i>Egr2</i> mice (red) (n = 4 and 5 mice for controls and <i>Crygn</i>^<i>Egr2</i>, respectively). No difference was observed for ABR thresholds in response to click stimuli (left) or noise burst stimuli (middle panel, n.s., not significant, p > 0.05). Threshold for pure tone stimuli were slightly but significantly better in <i>Crygn</i>^<i>Egr2</i> mice (<i>p</i> = 0.0279, 2-way ANOVA comparing the genotype). (<b>B</b>) Outer hair cell function measured by distortion product otoacoustic emission (DPOAE) growth function (left), maximal signal strength (middle, max. amplitude) and threshold for the 2f1-f2 distortion product (right). <i>Crygn</i>^<i>Egr2</i> mice had slightly improved 2f1-f2 distortion products indicated by increased amplitudes at 45 dB SPL stimulation (left, <i>p</i> = 0.1048, 2-way ANOVA) and small though non-significant improvements of DPOAE thresholds (right, <i>p</i> = 0.1013, 2-way ANOVA). This indicated intact outer hair cells and cochlear amplification in both genotypes. Arrows in left panel illustrate how threshold and amplitude were determined from the individual growth functions. (<b>C</b>) Processing of fast temporal modulation was measured by auditory steady state responses (ASSR) to amplitude modulated stimuli of increasing modulation speed (left, modulation frequency), as function of the modulation index (middle, modulation depth in %,) and for increasing level of the carrier (right, -20 to 60 dB hearing level, HL). For both genotypes, responses dropped for stimulation speeds above 1,024 Hz and were lost for faster modulation (2,048 Hz). Detection thresholds for modulated stimuli were at ca. 3–4% modulation, and response strength increased with carrier level. There was a tendency for <i>Crygn</i>^<i>Egr2</i> signals to level off at lower signal strength than the control (right panel, 55–60 dB hearing level), though this was not statistically significant. Insets schematically illustrate the used stimuli. (<b>D</b>) Changes of average peak amplitude for ABR wave I to IV (defined as peak to peak amplitudes, illustrated in the inset showing an example of an ABR recording with marked negative (n) and positive (p) peaks of either wave. The growth function shows significantly increased ABR amplitudes at wave IV at higher stimulus levels (> 50 dB, <i>p</i> < 0.001, 2-way ANOVA). Growth functions for ABR wave I and II amplitudes also were significantly changed, corroborating the results from the slightly improved ABR thresholds and DPOAE functions in <i>Crygn</i>^<i>Egr2</i> mice. Data for controls are shown as open bars, symbols and black lines, data for <i>Crygn</i>^<i>Egr2</i> mice are shown in red. Data represent mean and standard deviation (A,B) or standard error of the mean (C,D). The number of measured ears is indicated in each panel. n.s., not significant, *: p < 0.05, ***: p < 0.001, ****: p < 0.0001 in Holm-Sidak's multiple comparisons test. (*) indicates statistical results from uncorrected single comparison with <i>p</i> < 0.05 in 2-sided t-test.</p

Generation of a spatially restricted <i>Crygn</i> knockout mouse in the auditory hindbrain.

<p><b>(A)</b> Scheme of the knockout strategy, consisting of crossing a mouse line with a floxed allele of exon 2 of <i>Crygn</i> (<i>Crygn</i>:<i>tm1a</i> (EUCOMM)) and the <i>Egr2</i>::<i>Cre</i> driver line. Primers for probing recombination are depicted as black arrows. (<b>B)</b> Validation of the spatial ablation in the SOC. Left side: Genotyping of the floxed <i>Crygn</i> locus. In wt, a 524 bp long PCR product is amplified, whereas the mutant locus results in a 207 bp product. Right site: Confirmation of recombination in the SOC. Upon recombination, a 604 bp long product is amplified. The non-recombined locus is 3,411 bp in length and not amplified under the PCR conditions used. (<b>C)</b> RNA <i>in situ hybridization</i> analysis in the MNTB. An RNA probe complementary to exon 2 yields only signals in MNTB section of control mice whereas no signal is observed in <i>Crygn</i>^<i>Egr2</i> mice. In contrast, an RNA probe complementary to exons 2–4 still yields signals in the MNTB of <i>Crygn</i>^<i>Egr2</i> mice. This indicates transcription of the truncated <i>Crygn</i> gene. Scale bar is 200 μm.</p

Crygd/e-ir in the rat, mouse, and gerbil SOC at P4.

<p><b>(A)</b> Crygd/e labeling is observed in the rat MNTB and fibers of the acoustic stria. (<b>B)</b> LSO and MSO display moderate Cryge-ir. In the mouse, MNTB (<b>C</b>), LSO and MSO (<b>D</b>) are also labeled. (<b>E-F)</b> The gerbil MNTB, LSO and MSO show Crygd/e-ir similar to background. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.</p

Somatic copy number variant load in neurons of healthy controls and Alzheimer’s disease patients

Abstract The possible role of somatic copy number variations (CNVs) in Alzheimer’s disease (AD) aetiology has been controversial. Although cytogenetic studies suggested increased CNV loads in AD brains, a recent single-cell whole-genome sequencing (scWGS) experiment, studying frontal cortex brain samples, found no such evidence. Here we readdressed this issue using low-coverage scWGS on pyramidal neurons dissected via both laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) across five brain regions: entorhinal cortex, temporal cortex, hippocampal CA1, hippocampal CA3, and the cerebellum. Among reliably detected somatic CNVs identified in 1301 cells obtained from the brains of 13 AD patients and 7 healthy controls, deletions were more frequent compared to duplications. Interestingly, we observed slightly higher frequencies of CNV events in cells from AD compared to similar numbers of cells from controls (4.1% vs. 1.4%, or 0.9% vs. 0.7%, using different filtering approaches), although the differences were not statistically significant. On the technical aspects, we observed that LCM-isolated cells show higher within-cell read depth variation compared to cells isolated with FACS. To reduce within-cell read depth variation, we proposed a principal component analysis-based denoising approach that significantly improves signal-to-noise ratios. Lastly, we showed that LCM-isolated neurons in AD harbour slightly more read depth variability than neurons of controls, which might be related to the reported hyperploid profiles of some AD-affected neurons