Biology subjects

Manning, E. E.

Publications and source records attributed to Manning, E. E..

9 recordsLinked to original sources

bioRxiv2026

Modified RNA Extraction Methods to Eliminate Agarose Impurities in Precision-Cut Lung Slices

Precision-cut lung slices (PCLS) have emerged as a powerful tool for studying the biology of viable human lung tissue. However, the presence of agarose impurities compromises RNA yield and integrity during the extraction process. We tested whether using an alternative Plant kit RNA extraction method to wash agarose impurities or pre-dissolving agarose from PCLS implementing a dissolving buffer for routine RNA isolation in gel-electrophoresis would improve RNA quantity, quality, and integrity. Our results show that RNA quantity and integrity are highly compromised when using a conventional method of RNA extraction. The plant kit and dissolution of agarose increased the RNA quantity to 0.42{+/-}0.11 and 0.65{+/-}0.17 {micro}g/PCLS (measured by the Qubit) and integrity number to 6.60{+/-}0.59 and 9.13{+/-}0.39 (measured by the Bioanalyzer), respectively. The presence of impurities in conventional and Plant kit extractions misled to an overestimation of the RNA quantity and quality using the NanoDrop. The Plant kit and agarose dissolution showed a significant transcript integrity increase in GUSB (p<0.0001) and COL1A1 (p<0.05) expression, validating these methods over conventional extraction. We encourage laboratories applying PCLS experimentation to implement alternative methods to remove agarose impurities during RNA extraction, as well as to rely on sensitive quantitative techniques, such as the Qubit and Bioanalyzer, for RNA quantification and integrity measurements.

cell biology↗

bioRxiv2024

Dorsomedial striatal neuroinflammation causes excessive goal-directed action control by disrupting homeostatic astrocyte function

Compulsive actions are typically thought to reflect the dominance of habits over goal-directed action. To address this, we mimicked the striatal neuroinflammation that is frequently exhibited in individuals with compulsive disorders in rats, by injecting the endotoxin lipopolysaccharide into the posterior dorsomedial striatum, and assessed the consequences for behavioural control. Surprisingly, this manipulation caused rats to acquire and maintain goal-directed actions under conditions that would otherwise produce habits. Immunohistochemical analyses indicated that these behaviours were a result of astrocytic proliferation. To probe this further, we chemogenetically activated the Gi-pathway in striatal astrocytes, which altered the firing properties of nearby medium spiny neurons and modulated goal-directed action control. Together, results show that striatal neuroinflammation is sufficient to bias action selection toward excessive goal-directed control via dysregulated astrocyte function. If translatable, our findings suggest that, contrary to conventional views, individuals with striatal neuroinflammation might be more prone to maladaptive goal-directed actions than habits, and future interventions should aim to restore appropriate action control.

animal behavior and cognition↗

bioRxiv2024

Sapap3 knockout mice show threat bias under conflict during platform mediated avoidance task: implications for obsessive compulsive disorder

Obsessive compulsive disorder (OCD) typically involves cycling between symptoms of intrusive aversive thoughts (obsessions) and repetitive rituals aimed at avoiding these aversive outcomes (compulsions), which interferes with patients engagement with other important aspects of their lives. This cycling relationship between obsessions and compulsions highlights a potential role of impaired threat processing and avoidance behavior in OCD symptoms. The most effective behavioral therapy for OCD, exposure with response prevention (ERP), aims to break this cycle. However, it can be difficult for patients to access and engage with, suggesting a need for improved understanding of the neural mechanisms of threat processing and avoidance behavior in OCD to guide development of new and more effective treatments. Platform mediated avoidance (PMA) has proven to be a useful translational paradigm for use in rodents to examine avoidance neurobiology and models relevant to OCD, such as ERP and overtraining-induced persistent avoidance. However, to date this protocol has only been used in rats, and studies in transgenic mouse models relevant to OCD may shed further light on neural mechanisms relevant to disturbances in avoidance and threat processing in the disorder. To address this gap, we tested Sapap3 knockout (KO) mice, a leading preclinical model in OCD research, in the PMA task. Using this paradigm, we examined avoidance acquisition, expression, and extinction, as well as reward seeking under motivational conflict in two separate cohorts conditioned using higher (0.4 mA) or lower (0.24 mA) intensity shock. Surprisingly, the most striking difference observed in Sapap3-KOs vs control mice was heightened suppression of lever pressing for rewards during a tone signaling impending threat, suggesting a shift in action selection under motivational conflict (genotype effect avoidance conditioning: 0.24 mA cohort p=0.011, 0.4 mA cohort p=0.07; avoidance extinction: 0.24 mA p=0.057, 0.4 mA p=0.042). Avoidance responding was also acquired more slowly in Sapap3-KOs trained with a low intensity shock (time x genotype interaction p=0.025) and was extinguished more robustly following ERP (genotype effect p=0.043). In contrast, avoidance was similar between Sapap3 KOs and WT littermate controls trained using higher intensity shock (0.4 mA cohort time effect p<0.0001). Expression of the immediate early gene c-Fos associated with reinstatement of avoidance after ERP showed preliminary evidence for decreased activity of medial orbitofrontal cortex (mOFC) in KOs which may contribute to observed differences in PMA performance (KO vs WT mOFC c-Fos t-test p=0.0456). Together these findings suggest that mOFC dysfunction may contribute to increases in the influence of threats vs rewards over action selection in an animal model with relevance to OCD, and that the Sapap3-KO model presents valuable opportunities for deeper mechanistic investigation of avoidance and threat processing relevant to the human disorder.

neuroscience↗

bioRxiv2023

Optical Von-Frey method to determine nociceptive thresholds:a novel paradigm for preclinical pain assessment and analgesic screening

The goal of this study was to characterize a model that specifically activates peripheral nociceptors, allowing pure nociceptive thresholds to be monitored over a range of conditions including pathology or in screening analgesic compounds. Transgenic mice expressing channelrhodopsin-2 (ChR2) in cell populations positive for the transient receptor potential cation channel subfamily V member 1 (TRPV1) gene were bred to enable peripheral nociceptor photostimulation. Preliminary experiments confirmed the expected localisation pattern of ChR2 positive profiles in the dorsal root ganglion and superficial dorsal horn, mirroring TPRV1 expression. Brief hindpaw photostimulation with 470nm light caused hindpaw withdrawal and nocifensive behaviours in ChR2 positive animals but not control ChR2 negative animals. Using a simplified up/down approach, optical nociceptive thresholds were assessed with a 5-intensity hindpaw photostimulation paradigm, establishing the minimum intensity required to produce a withdrawal response (optical threshold). All testing was also video recorded and analysed post-hoc to assess additional photostimulation evoked behaviours. Repeated testing over several days showed optical nociceptive thresholds and response duration were similar, supporting the stability of these variables across a timeframe relevant to onset of pathology or drug administration. Optical nociceptive thresholds were also assessed following morphine administration (30 mg/kg), which significantly raised thresholds, highlighting analgesic screening utility of this model. Together, these findings demonstrate the peripheral photostimulation with optical thresholding is a useful addition to the preclinical nociception assessment toolkit, with the key advantage of inducing a purely nociceptive response to a non-invasive, non-tissue damaging stimulus.

neuroscience↗

bioRxiv2023

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma - DIPG), are uniformly fatal brain tumors that lack effective pharmacological treatment. Analysis of pooled CRISPR-Cas9 loss-of-function gene deletion screen datasets, identified PIK3CA and MTOR as targetable molecular dependencies across DIPG patient derived models, highlighting the therapeutic potential of the blood-brain barrier penetrant PI3K/Akt/mTOR inhibitor paxalisib. At the human equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic feedback resulting in increased blood glucose and insulin levels, commensurate with DIPG patients in Phase 1b clinical trials who experienced hyperglycemia/hyperinsulinemia. To exploit genetic dependences, but maintain compliance and benefit, we optimized a paxalisib treatment regimen that employed reduced dosing more frequently, in combination with the anti-hyperglycemic drug, metformin. Combining optimized dosing with metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending the survival of DIPG xenograft models. RNA sequencing and phosphoproteomic profiling of DIPG models treated with paxalisib identified increased calcium-activated PKC signaling. Using the brain penetrant PKC inhibitor, enzastaurin in combination with paxalisib, we synergistically extended the survival of orthotopic xenograft models, benefits further promoted by metformin; thus, identifying a clinically relevant DIPG combinatorial approach. Brief SummaryDiffuse intrinsic pontine glioma is a lethal childhood brain tumor. Here we identify PIK3CA as a genetic dependency targeted by the brain penetrant pan-PI3K-inhibitor paxalisib.

cancer biology↗

bioRxiv2023

Optogenetic recruitment of hypothalamic corticotrophin-releasing-hormone (CRH) neurons reduces motivational drive

Impaired motivational drive is a key feature of depression. Chronic stress is a known antecedent to the development of depression in humans and depressive-like states in animals. Whilst there is a clear relationship between stress and motivational drive, the mechanisms underpinning this association remain unclear. One hypothesis is that the endocrine system, via corticotropin-releasing hormone (CRH) in the paraventricular nucleus of the hypothalamus (PVN; PVNCRH), initiates a hormonal cascade resulting in glucocorticoid release, and that excessive glucocorticoids change brain circuit function to produce depression-related symptoms. Another, mostly unexplored hypothesis is that the direct activity of PVNCRH neurons and their input to other stress- and reward-related brain regions drives these behaviours. To further understand the direct involvement of PVNCRH neurons in motivation, we used optogenetic stimulation to activate these neurons one hour/day for 5 consecutive days and showed increased acute stress-related behaviours and long-lasting deficits in the motivational drive for sucrose. This was associated with increased Fos-protein expression in the lateral hypothalamus (LH). Direct stimulation of the PVNCRH inputs in the LH produced a similar pattern of effects on sucrose motivation. Together, these data suggest that PVNCRH neuronal activity may be directly responsible for changes in motivational drive and that these behavioural changes may, in part, be driven by PVNCRH synaptic projections to the LH.

neuroscience↗

bioRxiv2022

Hyperactivity of indirect pathway-projecting spiny projection neurons drives compulsive behavior

Compulsive behaviors are a hallmark symptom of obsessive compulsive disorder (OCD). Striatal hyperactivity has been linked to compulsive behavior generation in correlative studies in humans and causal studies in rodents. However, the contribution of the two distinct striatal output populations to the generation and treatment of compulsive behavior is unknown. These populations of direct and indirect pathway-projecting spiny projection neurons (SPNs) have classically been thought to promote or suppress actions, respectively, leading to a long-held hypothesis that increased output of direct relative to indirect pathway promotes compulsive behavior. Contrary to this hypothesis, here we find that indirect pathway hyperactivity drives compulsive grooming in the Sapap3-knockout mouse model of OCD-relevant behavior. Furthermore, we show that suppression of indirect pathway activity using optogenetics or treatment with the first-line OCD pharmacotherapy fluoxetine is associated with reduced compulsive behavior. Together, these findings highlight the striatal indirect pathway as a potential new treatment target for compulsive behavior.

neuroscience↗

bioRxiv2021

Independent and distinct patterns of abnormal lateral orbitofrontal cortex activity during compulsive grooming and reversal learning normalize after fluoxetine

BackgroundPatients with obsessive-compulsive disorder (OCD) display disrupted performance and abnormal lateral orbitofrontal cortex (LOFC) activity during reversal learning tasks, yet it is unknown whether compulsions and reversal learning deficits share a common neural substrate. To answer this question, we measured neural activity with in vivo calcium imaging in LOFC during compulsive grooming and reversal learning before and after fluoxetine treatment. MethodsSapap3-knockout (KO) mice were used as a model for OCD-relevant behaviors. Sapap3-KOs and control littermates were injected with virus encoding GCaMP6f and implanted with gradient-index lenses to visualize LOFC activity using miniature microscopes. Grooming, reversal learning, and neural activity were measured pre- and post-fluoxetine treatment (18mg/kg, 4 weeks). ResultsBaseline compulsive grooming and reversal learning impairments in KOs improved after fluoxetine treatment. Additionally, KOs display distinct patterns of abnormal LOFC activity during grooming and reversal learning, both of which normalize after fluoxetine. Finally, modulation in response to reversal learning and compulsive behavior are independent, as reversal learning-associated neurons are distributed randomly amongst grooming-associated neurons (i.e. overlap is what would be expected by chance). ConclusionsIn OCD, the LOFC is disrupted during both compulsive behaviors and reversal learning, yet whether these behaviors share common neural underpinnings is unknown. We find that the LOFC plays distinct and independent roles in compulsive grooming and impaired reversal learning and their improvement with fluoxetine. These findings suggest that LOFC plays separate roles in pathophysiology and treatment of different perseverative behaviors in OCD.

neuroscience↗

bioRxiv2019

Disruption of prepulse inhibition is associated with severity of compulsive behavior and nucleus accumbens dopamine system changes in Sapap3 knockout mice

Obsessive compulsive disorder (OCD) is associated with disruption of sensorimotor gating, which may contribute to difficulties inhibiting intrusive thoughts and compulsive rituals. Neural mechanisms underlying these disturbances are unclear; however, striatal dopamine is implicated in regulation of sensorimotor gating and OCD pathophysiology. The goal of this study was to examine the relationships between sensorimotor gating, compulsive behavior, and striatal dopamine receptor levels in Sapap3 knockout mice (KOs), a widely used preclinical model system for OCD research. We found a trend for disruption of sensorimotor gating in Sapap3-KOs using the translational measure prepulse inhibition (PPI); however, there was significant heterogeneity in both PPI and compulsive grooming in KOs. Disruption of PPI was significantly correlated with a more severe compulsive phenotype. In addition, PPI disruption and compulsive grooming severity were associated with reduced dopamine D1 and D2/3 receptor density in the nucleus accumbens core (NAcC). Compulsive grooming progressively worsened in Sapap3-KOs tested longitudinally, but PPI disruption was first detected in high-grooming KOs at 7 months of age. Through detailed characterization of individual differences in OCD-relevant behavioral and neurochemical measures, our findings suggest that NAcC dopamine receptor changes may be involved in disruption of sensorimotor gating and compulsive behavior relevant to OCD.

neuroscience↗