Biology subjects

Walters, B. J.

Publications and source records attributed to Walters, B. J..

6 recordsLinked to original sources

bioRxiv2026

The RNA editing enzyme ADARB1 is readily detectable in primary auditory neurons and provides a means for automated counting

Spiral ganglion neurons (SGNs) are the primary auditory afferents in the inner ear. These neurons degenerate in response to a number of conditions, including auditory neuropathies, concussions, and aging. Research to assess the extent of degeneration and to test the efficacy of protective or rehabilitative strategies requires quantification of SGNs from tissue sections. However, manual counting of SGNs can be arduous and time-consuming due to dense crowding and the lack of reliable nuclear-specific labels. SGNs receive afferent input via GluA2-containing AMPA receptors. As the Gria2 transcripts that code for GluA2 must undergo RNA editing to ensure calcium impermeability, we hypothesized that SGNs would express high levels of the adenosine deaminase acting on RNA (ADAR) enzyme ADARB1. Here we confirm enriched expression of Adarb1 in SGNs via in situ hybridization and show that anti-ADARB1 antibodies robustly label the nuclei of both type I and type II SGNs in cochlear sections from young and aged mice. Neuronal specificity was confirmed using antibodies against neurofilament heavy chain (NFH), human antigen D (HuD), GATA binding protein 3 (GATA3), and SRY-box 2 (SOX2). A blinded investigator manually counted SGNs via NFH staining, and these were compared to automated counts of ADARB1-positive nuclei using the analyze particles function in ImageJ. A concordance correlation coefficient and Bland-Altman analysis demonstrated strong agreement between the manual and automated counts. Additionally, immunolabeling of ADARB1 in macaque and human temporal bone sections confirm robust labeling of SGN nuclei, suggesting broad utility of ADARB1 immunolabeling for automated counts of SGNs across species.

neuroscience↗

bioRxiv2025

Opposing effects of H2A.Z on memory, transcription and pathology in male and female Alzheimer's disease mice and patients

Alzheimers disease (AD) is a devastating neurodegenerative disease that disproportionately impacts women, but underlying mechanisms for sex-divergent outcomes are unknown. Here, we show that the histone variant H2A.Z is a novel sex-specific regulator of AD in human patients and AD model mice. Specifically, H2A.Z binding in chromatin declines in female and increases in male AD patients, indicating opposite patterns of AD-related H2A.Z dysregulation each sex. These sex differences were recapitulated in the 5xFAD model of AD, in which females accumulated H2A.Z at early disease stages and lost H2A.Z as the disease progressed, suggesting that H2A.Z occupancy shifts with advancing disease. Males showed no change in H2A.Z binding in early disease, but exhibited increased binding as disease progressed, albeit to a lesser extent than females. Consistent with sex-specific H2A.Z dysregulation, H2A.Z depletion produced sex-specific changes in gene expression, whereby H2A.Z was more repressive in female than in male mice and in 5xFAD than in WT males, suggesting that H2A.Zs role in transcription varies with sex and disease. Moreover, H2A.Z depletion improved memory and AD pathology in females, while impairing memory and worsening pathology in male mice. Together, these data suggest that H2A.Z is protective in males and detrimental in females with AD, with key implications for sex-specific therapeutic targeting of chromatin factors.

neuroscience↗

bioRxiv2024

PCP Auto Count: A Novel Fiji/ImageJ plug-in for automated quantification of planar cell polarity and cell counting

BackgroundDuring development, planes of cells give rise to complex tissues and organs. The proper functioning of these tissues is critically dependent on proper inter- and intra-cellular spatial orientation, a feature known as planar cell polarity (PCP). To study the genetic and environmental factors affecting planar cell polarity investigators must often manually measure cell orientations, which is a time-consuming endeavor. MethodologyTo automate cell counting and planar cell polarity data collection we developed a Fiji/ImageJ plug-in called PCP Auto Count (PCPA). PCPA analyzes binary images and identifies "chunks" of white pixels that contain "caves" of infiltrated black pixels. Inner ear sensory epithelia including cochleae (P4) and utricles (E17.5) from mice were immunostained for {beta}II-spectrin and imaged on a confocal microscope. Images were preprocessed using existing Fiji functionality to enhance contrast, make binary, and reduce noise. An investigator rated PCPA cochlear angle measurements for accuracy using a 1-5 agreement scale. For utricle samples, we directly compared PCPA derived measurements against manually derived angle measurements using concordance correlation coefficients (CCC) and Bland-Altman limits of agreement. Finally, PCPA was tested against a variety of images copied from publications examining PCP in various tissues and across various species. ResultsPCPA was able to recognize and count 99.81% of cochlear hair cells (n = 1,1541 hair cells) in a sample set, and was able to obtain ideally accurate planar cell polarity measurements for over 96% of hair cells. When allowing for a <10{degrees} deviation from "perfect" measurements, PCPAs accuracy increased to >98%. When manual angle measurements for E17.5 utricles were compared, PCPAs measurements fell within -9 to +10 degrees of manually obtained mean angle measures with a CCC of 0.999. Qualitative examination of example images of Drosophila ommatidia, mouse ependymal cells, and mouse radial progenitors revealed a high level of accuracy for PCPA across a variety of stains, tissue types, and species. Altogether, the data suggest that the PCPA plug-in suite is a robust and accurate tool for the automated collection of cell counts and PCP angle measurements.

developmental biology↗

bioRxiv2023

Large-scale annotated dataset for cochlear hair cell detection and classification

Our sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 90,000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

neuroscience↗

bioRxiv2022

Identification of putative GATA3 regulatory elements and comparison of GATA3 distribution in cochleae of mice, rats, macaques, and humans.

The transcription factor GATA3 plays a critical role in the development of neurons and sensory epithelia of the inner ear. In mouse cochleae, GATA3 is downregulated in certain supporting cells (SCs) and in type I spiral ganglion neurons (SGNs) after development. This reduction of GATA3 in SCs severely limits Atoh1-induced hair cell (HC) regeneration and suggests that a similar downregulation in human cochleae may be limiting for regenerative therapies. However, it is unknown whether GATA3 is similarly or differentially regulated in primates versus rodents. Using CAGE-seq data, we compared over 40 putative GATA3 regulatory elements across species and found both conserved and non-conserved sequences. To assess whether cochlear GATA3 distribution is similar or different between rodents and primates, we immunostained cochleae from mice, rats, macaques, and humans using antibodies raised against highly conserved GATA3 peptide sequences. GATA3 immunostaining in the organs of Corti from all four species revealed a large degree of conservation, where SCs medial and lateral to cochlear HCs exhibited robust nuclear GATA3 immunolabeling, but pillar and Deiters cells had significantly reduced GATA3 immunoreactivity. In all four species, GATA3 was expressed in a subset of SGNs that largely co-expressed peripherin suggesting they were type II SGNs. Only one difference emerged, wherein human cochlear inner hair cells were not GATA3 immunoreactive despite being so in the other species. Overall, the pattern of GATA3 expression in primates appears similar to rodents and reinforces the notion that ATOH1 mediated regenerative therapies may be limited by reduced GATA3 expression in adult SCs.

molecular biology↗

bioRxiv2021

Off-target expression of Cre-dependent adeno-associated viruses in wild type C57BL/6J mice

Adeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although non-amplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the hippocampus and cortex of wild-type C57BL/6J mice. While we observed weak but consistent non-amplified off-target DIO expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed extensive Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype or promoter. We also evaluated whether Cre-independent expression had functional effects via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). The DREADD agonist C21 had no effect on contextual fear conditioning or cFos expression in DIO-hM3Dq-mCherry+ cells of C57BL/6J mice. Taken together, our results indicate that DIO constructs have considerable off-target expression in wild type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression. Significance StatementAdeno-associated viruses (AAV) are widely used in neuroscience because of their safety and ease of use. Combined with specific promoters, Cre/loxP, and stereotaxic injections, highly specific targeting of cells and circuits within the brain can be achieved. In the present study we injected Cre-dependent AAVs into wild-type C57BL/6J mice and found considerable Cre-independent viral expression of AAVs encoding mCherry, GFP, or hM3Dq following immunohistochemical amplification of the fluorescent reporter protein. Importantly, we observed no functional effects of the Cre-independent expression in the hippocampus, as C21 had no detectable effect on DIO-hM3Dq-mCherry infected neurons in C57BL/6J mice. Given the widespread use of DIO rAAVs by the neuroscience community, our data supports careful consideration when using DIO constructs in control animals.

neuroscience↗