Biology subjects

Sharp, P.

Publications and source records attributed to Sharp, P..

9 recordsLinked to original sources

bioRxiv2024

Etomidate as an Effective Alternative Method of Euthanizing Adult Zebrafish (Danio Rerio)

We assessed the efficacy of etomidate in euthanizing adult zebrafish. The aims of this study were to investigate: 1) the efficacy of 2 concentrations and 2) the effects at 2 densities. Aim 1-Fish (n=10 fish/group) were immersed in either 6 mg/L (Eto-6) or 10 mg/L (Eto-10) etomidate. Aim 2- either 5 or 10 fish were immersed in Eto-6. Tanks were video recorded and scored by two blinded observers. The parameters monitored included: 1) loss of righting reflex (LORR); 2) undulation cessation; 3) cessation of operculation (COO); and 4) loss of startle reflex. After the 30 minute immersion, fish were transferred to recovery tanks and monitored once every 5 minutes for 60 minutes. Results - Aim 1, the time to undulation cessation, COO, and loss of startle reflex was significantly shorter in the Eto-10 group and there was no difference in LORR. Aim 2, the time to LORR and undulation cessation was significantly shorter in the 5 fish/L group and there was no significant difference in time to COO or loss of startle reflex. No aversive behaviors were noted and fish in all groups were effectively euthanized by 30 minutes. This study indicates that Eto-6 or Eto-10 effectively euthanizes adult zebrafish.

animal behavior and cognition↗

bioRxiv2023

Improved modeling of RNA-binding protein motifs in an interpretable neural model of RNA splicing

Sequence-specific RNA-binding proteins (RBPs) play central roles in splicing decisions, but their exact binding locations and activities are difficult to predict. Here, we describe a modular splicing architecture that leverages in vitro-derived RNA affinity models for 79 human RBPs and the annotated human genome to produce improved models of RBP binding and activity. Binding and activity are modeled by separate Motif and Aggregator components that can be mixed and matched, enforcing sparsity to improve interpretability. Standard affinity models yielded reasonable predictions, but substantial improvements resulted from using a new Adjusted Motif (AM) architecture. While maintaining accurate modeling of in vitro binding, training these AMs on the splicing task yielded improved predictions of binding sites in vivo and of splicing activity, using independent crosslinking and massively parallel splicing reporter assay data. The modular structure of our model enables improved generalizability to other species (insects, plants) and to exons of different evolutionary ages.

genomics↗

bioRxiv2021

The effects of locomotion on sensory-evoked haemodynamic responses in the cortex of awake mice

Investigating neurovascular coupling in awake rodents is becoming ever more popular due, in part, to our increasing knowledge of the profound impacts that anaesthesia can have upon brain physiology. Although awake imaging brings with it many advantages, we still do not fully understand how voluntary locomotion during imaging affects sensory-evoked haemodynamic responses. In this study we investigated how evoked haemodynamic responses can be affected by the amount and timing of locomotion. Using an awake imaging set up, we used 2D-Optical Imaging Spectroscopy (2D-OIS) to measure changes in cerebral haemodynamics within the sensory cortex of the brain during either 2s whisker stimulation or spontaneous (no whisker stimulation) experiments, whilst animals could walk on a spherical treadmill. We show that locomotion alters haemodynamic responses. The amount and timing of locomotion relative to whisker stimulation is important, and can significantly impact sensory-evoked haemodynamic responses. If locomotion occurred before or during whisker stimulation, the amplitude of the stimulus-evoked haemodynamic response was significantly altered. Therefore, monitoring of locomotion during awake imaging is necessary to ensure that conclusions based on comparisons of evoked haemodynamic responses (e.g., between control and disease groups) are not confounded by the effects of locomotion.

neuroscience↗

bioRxiv2020

Assessment of Neurovascular Coupling & Cortical Spreading Depression in Mixed Models of Atherosclerosis & Alzheimer's Disease

Neurovascular coupling is a critical brain mechanism whereby changes to blood flow accompany localised neural activity. The breakdown of neurovascular coupling is linked to the development and progression of several neurological conditions including dementia. In this study, we examined cortical haemodynamics in preparations that modelled Alzheimers disease (J20-AD) and atherosclerosis (PCSK9-ATH) between 9-12m of age. We report novel findings with atherosclerosis where neurovascular decline is characterised by significantly reduced blood volume, levels of oxyhaemoglobin & deoxyhaemoglobin, in addition to global neuroinflammation. In the comorbid mixed model (J20-PCSK9-MIX), we report a 3x fold increase in hippocampal amyloid-beta plaques. A key finding was that cortical spreading depression (CSD) due to electrode insertion into the brain was worse in the diseased animals and led to a prolonged period of hypoxia. These findings suggest that systemic atherosclerosis can be detrimental to neurovascular health and that having cardiovascular comorbidities can exacerbate pre-existing Alzheimers-related amyloid-plaques.

neuroscience↗

bioRxiv2019

Enhanced Cerebral Blood Volume under Normobaric Hyperoxia in the J20-hAPP Mouse Model of Alzheimer's Disease

Early impairments to neurovascular coupling have been proposed to be a key pathogenic factor in the onset and progression of Alzheimers disease (AD). Studies have shown impaired neurovascular function in several mouse models of AD, including the J20-hAPP mouse. In this study, we aimed to investigate early neurovascular changes using wild-type (WT) controls and J20-hAPP mice at 6-9 months of age, by measuring cerebral haemodynamics and neural activity to physiological sensory stimulations. A thinned cranial window was prepared to allow access to cortical vasculature and imaged using 2D-optical imaging spectroscopy (2D-OIS). After chronic imaging sessions where the skull was intact, a terminal acute imaging session was performed where an electrode was inserted into the brain to record simultaneous neural activity. We found that cerebral haemodynamic changes were significantly enhanced in J20-hAPP mice compared with controls in response to physiological stimulations, potentially due to the significantly higher neural activity (hyperexcitability) seen in the J20-hAPP mice. Thus, neurovascular coupling remained preserved under a chronic imaging preparation. Further, under hyperoxia, the baseline blood volume and saturation of all vascular compartments in the brains of J20-hAPP mice were substantially enhanced compared to WT controls, but this effect disappeared under normoxic conditions. This study highlights novel findings not previously seen in the J20-hAPP mouse model, and may point towards a potential therapeutic strategy by driving an increased baseline blood flow to the brain, thereby potentially enhancing the clearance of beta-amyloid.

neuroscience↗

bioRxiv2019

Key aspects of neurovascular control mediated by specific populations of inhibitory cortical interneurons

Inhibitory interneurons can evoke vasodilation and vasoconstriction, making them potential cellular drivers of neurovascular coupling. However, the specific regulatory roles played by particular interneuron subpopulations remain unclear. Our purpose was therefore to adopt a cell-specific optogenetic approach to investigate how somatostatin (SST) and neuronal nitric oxide synthase (NOS1)-expressing interneurons might influence neurovascular relationships. In mice, specific activation of SST- or NOS1-interneurons was sufficient to evoke haemodynamic changes similar to those evoked by physiological whisker stimulation. In the case of NOS1-interneurons, robust haemodynamic changes occurred with minimal changes in neural activity. Conversely, activation of SST-interneurons produced robust changes in evoked neural activity with shallow cortical excitation and pronounced deep layer cortical inhibition. This often resulted in a central increase in blood volume with corresponding surround decrease, analogous to the negative BOLD signal. These results demonstrate the role of specific populations of cortical interneurons in the active control of neurovascular function.

neuroscience↗

bioRxiv2018

The Time Course of Recognition Memory Impairment and Glial Pathology in the hApp-J20 Mouse Model of Alzheimer’s Disease

The role of cellular changes in the neurovascular unit is increasingly being investigated to understand the pathogenesis of Alzheimers disease. The aim of the current study was to determine the time course of recognition memory impairment in the J20 mouse model of AD, in relation to neuroinflammatory responses and the pathology of A{beta}.\n\nMale hAPP-J20 and wild-type mice were assessed at 3, 6, 9, and 12 months of age. The spontaneous object recognition (SOR) task provided a measure of memory, with assessment of both a short delay (1 min) and a long delay (4 hrs). Immunohistochemistry was used to characterise A{beta}-deposition, and quantify astrocyte and microglial responses.\n\nAt all ages tested J20 mice had impaired long-term, but preserved short-term, recognition memory. Wild-types demonstrated preserved long-term memory up to 9 months of age, and preserved short-term memory at all ages tested. Plaque pathology in the J20 mice was present from 6 months onwards, with co-localisation of reactive microglia and activated astrocytes. Reactive microglia and astrocyte activation in the hippocampus were significantly greater in the J20 mice at 9 months, compared to wild-types.\n\nThis study contributes to our understanding of the pathological and cognitive mechanisms at play in AD. J20 mice showed impairment in retaining information over longer periods from an early age, preceding the deposition of A{beta} and glial activation. Defining early physiological changes in relation to cognitive decline could provide insight into new therapeutic targets early in the disease process, when intervention is most likely to effectively slow disease progression.

neuroscience↗

bioRxiv2017

Direct in vivo mapping of functional suppressors in glioblastoma genome

Glioblastoma (GBM) is one of the deadliest cancers, with limited effective treatments and single-digit five-year survival 1-7. A causative understanding of genetic factors that regulate GBM formation is of central importance 8-19. However, a global, quantitative and functional understanding of gliomagenesis in the native brain environment has been lacking due to multiple challenges. Here, we developed an adeno-associated virus (AAV) mediated autochthonous CRISPR screen and directly mapped functional suppressors in the GBM genome. Stereotaxic delivery of an AAV library targeting significantly mutated genes into fully immunocompetent conditional Cas9 mice robustly led to gliomagenesis, resulting in tumors that recapitulate features of human GBM. Targeted capture sequencing revealed deep mutational profiles with diverse patterns across mice, uncovering in vivo roles of previously uncharacterized factors in GBM such as immune regulator B2m, zinc finger protein Zc3h13, transcription repressor Cic, epigenetic regulators Mll2/3 and Arid1b, alongside canonical tumor suppressors Nf1 and Pten. Comparative cancer genomics showed that the mutation frequencies across all genes tested in mice significantly correlate with those in human from two independent patient cohorts. Co-mutation analysis identified frequently co-occurring driver combinations, which were validated using AAV minipools, such as Mll2, B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1. Distinct from Nf1-oncotype tumors, Rb1-oncotype tumors exhibit undifferentiated histopathology phenotype and aberrant activation of developmental reprogramming signatures such as Homeobox gene clusters. The secondary addition of Zc3h13 or Pten mutations drastically altered the gene expression profiles of Rb1 mutants and rendered them more resistant to the GBM chemotherapeutic temozolomide. Our study provides a systematic functional landscape of GBM suppressors directly in vivo, opening new paths for high-throughput molecular mapping and cancer phenotyping.

systems biology↗

bioRxiv2017

Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing

High-resolution, high-throughput direct in vivo screening of functional genetic factors in native tissues has long been challenging. Adeno-associated viruses (AAV) are powerful carriers of transgenes and have been shown to mediate efficient genome editing in various organs in mice. Here, we developed a new technological approach, Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing (PASTAS), and demonstrated its application for directly mapping functional cancer driver variants in the mouse liver in an autochthonous manner. Intravenous delivery of an AAV-CRISPR library targeting a set of the most frequently mutated tumor suppressor genes into fully immunocompetent conditional Cas9 knock-in mice consistently generated highly complex autochthonous liver tumors. The molecular landscapes of these genetically diverse tumors were mapped out by deep direct readout of Cas9-generated variants at predicted sgRNA cut sites using molecular inversion probe sequencing. Co-occurrence and correlation analyses as well as validation with lower complexity minipools further confirmed the potency of various co-mutated drivers. The PASTAS method can be applied to virtually any gene sets, any cancer types, or any type of in vivo genetic studies other than cancer.

systems biology↗