Neuropilin-2 as a Novel Angiogenic Player: deciphering the contributions of neuropilin-2 during developmental and pathological angiogenesis

Angiogenesis, the growth of neovasculature from pre-existing vessels, is driven by the stimulation of endothelial cells (ECs) by pro-angiogenic factors to invade the surrounding tissue and expand the vascular network. The extracellular matrix (ECM) component fibronectin (FN), and its canonical receptors, α5β1 and αvβ3 integrins are upregulated on angiogenic vasculature. Historically, the selective targeting of these FN receptors was postulated to provide directed anti-angiogenic therapy against pathologies typified by uncontrolled vascular growth such as cancer and retinopathy. Genetic ablation of these integrins however, proved not to inhibit the development of excessive vascularisation, and in some cases even accelerated the pathological phenotype. It is now believed that the failure of such integrin-based therapies can be attributed to the action of VEGF co-receptors, such as neuropilin-1 (NRP1) and neuropilin-2 (NRP2), to rescue and promote a pro-angiogenic phenotype. Unlike NRP1, far less is known about NRP2's role in microvascular ECs during angiogenesis, and therefore we sought to characterise NRP2's interaction with α5β1 integrin in ECs and investigate whether a dual contribution with NRP1 exists to promote developmental and pathological angiogenesis. To achieve this, we implemented siRNA-mediated depletion in vitro, and developed and employed genetically modified mouse models (GEMMs) in vivo to study the angiogenic consequences of inducing EC specific deletion of NRP2 alone, or in tandem with α5 integrin or NRP1. Our results indicate that NRP2 promotes polarised EC migration and adhesion to FN by directing the cellular trafficking of α5 integrin-phospho-focal adhesion kinase (p-FAK) complexes in a Rab11-dependent manner. By utilising both physiological and pathological models of angiogenesis, we also provide evidence that NRP2 promotes the formation of stable cell-matrix interactions to facilitate tip cell sprouting in the postnatal retina, and is essential for tumour vascularisation. In addition, by targeting both endothelial NRPs we were able to successfully arrest tumour angiogenesis completely

Pseudoneglect and visual attention networks

Pseudoneglect represents the tendency for healthy individuals to show a slight but consistent bias in favour of stimuli appearing in the left visual field. The bias is often measured using variants of the line bisection task. An accurate model of the functional architecture of the visuospatial attention system must account for this widely observed phenomenon, as well as for modulation of the direction and magnitude of the bias within individuals by a variety of factors relating to the state of the participant and/or stimulus characteristics. To date, the neural correlates of pseudoneglect remain relatively unmapped. In the current thesis, I employed a combination of psychophysical measurements, electroencephalography (EEG) recording and transcranial direct current stimulation (tDCS) in an attempt to probe the neural generator(s) of pseudoneglect. In particular, I wished to utilise and investigate some of the factors known to modulate the bias (including age, time-on-task and the length of the to-be-bisected line) in order to identify neural processes and activity that are necessary and sufficient for the lateralized bias to arise. Across four experiments utilising a computerized version of a perceptual line bisection task, pseudoneglect was consistently observed at baseline in healthy young participants. However, decreased line length (experiments 1, 2 and 3), time-on-task (experiment 1) and healthy aging (experiment 3) were all found to modulate the bias. Specifically, all three modulations induced a rightward shift in subjective midpoint estimation. Additionally, the line length and time-on-task effects (experiment 1) and the line length and aging effects (experiment 3) were found to have additive relationships. In experiment 2, EEG measurements revealed the line length effect to be reflected in neural activity 100 – 200ms post-stimulus onset over source estimated posterior regions of the right hemisphere (RH: temporo-parietal junction (TPJ)). Long lines induced a hemispheric asymmetry in processing (in favour of the RH) during this period that was absent in short lines. In experiment 4, bi-parietal tDCS (Left Anodal/Right Cathodal) induced a polarity-specific rightward shift in bias, highlighting the crucial role played by parietal cortex in the genesis of pseudoneglect. The opposite polarity (Left Cathodal/Right Anodal) did not induce a change in bias. The combined results from the four experiments of the current thesis provide converging evidence as to the crucial role played by the RH in the genesis of pseudoneglect and in the processing of visual input more generally. The reduction in pseudoneglect with decreased line length, increased time-on-task and healthy aging may be explained by a reduction in RH function, and hence contribution to task processing, induced by each of these modulations. I discuss how behavioural and neuroimaging studies of pseudoneglect (and its various modulators) can provide empirical data upon which accurate formal models of visuospatial attention networks may be based and further tested

Low pre-stimulus EEG alpha power amplifies visual awareness but not visual sensitivity

Pre‐stimulus oscillatory neural activity has been linked to the level of awareness of sensory stimuli. More specifically, the power of low frequency oscillations (primarily in the alpha‐band, i.e. 8‐14 Hz) prior to stimulus‐onset is inversely related to measures of subjective performance in visual tasks, such as confidence and visual awareness. Intriguingly, the same EEG‐signature does not seem to influence objective measures of task performance (i.e. accuracy). We here examined whether this dissociation holds when stringent accuracy measures are used. Previous EEG‐studies have employed 2‐alternative forced‐choice (2‐AFC) discrimination tasks to link pre‐stimulus oscillatory activity to correct/incorrect responses as an index of accuracy/objective performance at the single‐trial level. However, 2‐AFC tasks do not provide a good estimate of single‐trial accuracy, as many of the responses classified as correct will be contaminated by guesses (with the chance correct response rate being 50%). Here instead, we employed a 19‐AFC letter identification task to measure accuracy and the subjectively reported level of perceptual awareness on each trial. As the correct guess rate is negligible (~5%), this task provides a purer measure of accuracy. Our results replicate the inverse relationship between pre‐stimulus alpha/beta‐band power and perceptual awareness ratings in the absence of a link to discrimination accuracy. Pre‐stimulus oscillatory phase did not predict either subjective awareness or accuracy. Our results hence confirm a dissociation of the pre‐stimulus EEG power ‐ task performance link for subjective versus objective measures of performance, and further substantiate pre‐stimulus alpha power as a neural predictor of visual awareness

Information search under uncertainty across transdiagnostic psychopathology and healthy ageing

When making decisions in everyday life, we often rely on an internally generated sense of confidence to help us revise and direct future behaviours. For instance, confidence directly informs whether further information should be sought prior to commitment to a final decision. Many studies have shown that aging and both clinical and sub-clinical symptoms of psychopathology are associated with systematic alterations in confidence. However, it remains unknown whether these confidence distortions influence information-seeking behaviour. We investigated this question in a large general population sample (N = 908). Participants completed a battery of psychiatric symptom questionnaires and performed a perceptual decision-making task with confidence ratings in which they were offered the option to seek helpful information (at a cost) before committing to a final decision. Replicating previous findings, an 'anxious-depression' (AD) symptom dimension was associated with systematically low confidence, despite no detriment in objective task accuracy. Conversely, a 'compulsive behaviour and intrusive thoughts' (CIT) dimension was associated with impaired task accuracy but paradoxical over-confidence. However, neither symptom dimension was significantly associated with an increased or decreased tendency to seek information. Hence, participants scoring highly for AD or CIT did not use the option to information seek any more than average to either increase their confidence (AD) or improve the accuracy of their decisions (CIT). In contrast, older age was associated with impaired accuracy and decreased confidence initially, but increased information seeking behaviour mediated increases in both accuracy and confidence for final decisions. Hence, older adults used the information seeking option to overcome initial deficits in objective performance and to increase their confidence accordingly. The results show an appropriate use of information seeking to overcome perceptual deficits and low confidence in healthy aging which was not present in transdiagnostic psychopathology.</p

No interaction between tDCS current strength and baseline performance: a conceptual replication

Several recent studies have reported non-linear effects of transcranial direct current stimulation (tDCS), which has been attributed to an interaction between the stimulation parameters (e.g., current strength, duration) and the neural state of the cortex being stimulated (e.g., indexed by baseline performance ability, age) (see Fertonani and Miniussi, 2016). We have recently described one such non-linear interaction between current strength and baseline performance on a visuospatial attention (landmark) task (Benwell et al., 2015). In this previous study, we induced a small overall rightward shift of spatial attention across 38 participants using bi-hemispheric tDCS applied for 20 min (concurrent left posterior parietal (P5) anode and right posterior parietal (P6) cathode) relative to a sham protocol. Importantly, this shift in bias was driven by a state-dependent interaction between current intensity and the discrimination sensitivity of the participant at baseline (pre-stimulation) for the landmark task. Individuals with high discrimination sensitivity (HDS) shifted rightward in response to low- (1 mA) but not high-intensity (2 mA) tDCS, whereas individuals with low discrimination sensitivity (LDS) shifted rightward with high- but not low-intensity stimulation. However, in Benwell et al. (2015) current strength was applied as a between-groups factor, where half of the participants received 1 mA and half received 2 mA tDCS, thus we were unable to compare high and low-intensity tDCS directly within each individual. Here we aimed to replicate these findings using a within-group design. Thirty young adults received 15 min of 1 and 2 mA tDCS, and a sham protocol, each on different days, to test the concept of an interaction between baseline performance and current strength. We found no overall rightward shift of spatial attention with either current strength, and no interaction between performance and current strength. These results provide further evidence of low replicability of non-invasive brain stimulation protocols, and the need for further attempts to replicate the key experimental findings within this field

Trial-by-trial co-variation of pre-stimulus EEG alpha power and visuospatial bias reflects a mixture of stochastic and deterministic effects

Human perception of perithreshold stimuli critically depends on oscillatory EEG activity prior to stimulus onset. However, it remains unclear exactly which aspects of perception are shaped by this pre‐stimulus activity and what role stochastic (trial‐by‐trial) variability plays in driving these relationships. We employed a novel jackknife approach to link single‐trial variability in oscillatory activity to psychometric measures from a task that requires judgement of the relative length of two line segments (the landmark task). The results provide evidence that pre‐stimulus alpha fluctuations influence perceptual bias. Importantly, a mediation analysis showed that this relationship is partially driven by long‐term (deterministic) alpha changes over time, highlighting the need to account for sources of trial‐by‐trial variability when interpreting EEG predictors of perception. These results provide fundamental insight into the nature of the effects of ongoing oscillatory activity on perception. The jackknife approach we implemented may serve to identify and investigate neural signatures of perceptual relevance in more detail

Psychiatrically relevant signatures of domain-general decision-making and metacognition in the general population

Human behaviours are guided by how confident we feel in our abilities. When confidence does not reflect objective performance, this can impact critical adaptive functions and impair life quality. Distorted decision-making and confidence have been associated with mental health problems. Here, utilising advances in computational and transdiagnostic psychiatry, we sought to map relationships between psychopathology and both decision-making and confidence in the general population across two online studies (N’s = 344 and 473, respectively). The results revealed dissociable decision-making and confidence signatures related to distinct symptom dimensions. A dimension characterised by compulsivity and intrusive thoughts was found to be associated with reduced objective accuracy but, paradoxically, increased absolute confidence, whereas a dimension characterized by anxiety and depression was associated with systematically low confidence in the absence of impairments in objective accuracy. These relationships replicated across both studies and distinct cognitive domains (perception and general knowledge), suggesting that they are reliable and domain general. Additionally, whereas Big-5 personality traits also predicted objective task performance, only symptom dimensions related to subjective confidence. Domain-general signatures of decision-making and metacognition characterise distinct psychological dispositions and psychopathology in the general population and implicate confidence as a central component of mental health

Stimulus-Driven Brain Rhythms within the Alpha Band: The Attentional-Modulation Conundrum

Two largely independent research lines use rhythmic sensory stimulation to study visual processing. Despite the use of strikingly similar experimental paradigms, they differ crucially in their notion of the stimulus-driven periodic brain responses: one regards them mostly as synchronized (entrained) intrinsic brain rhythms; the other assumes they are predominantly evoked responses [classically termed steady-state responses (SSRs)] that add to the ongoing brain activity. This conceptual difference can produce contradictory predictions about, and interpretations of, experimental outcomes. The effect of spatial attention on brain rhythms in the alpha band (8–13 Hz) is one such instance: alpha-range SSRs have typically been found to increase in power when participants focus their spatial attention on laterally presented stimuli, in line with a gain control of the visual evoked response. In nearly identical experiments, retinotopic decreases in entrained alpha-band power have been reported, in line with the inhibitory function of intrinsic alpha. Here we reconcile these contradictory findings by showing that they result from a small but far-reaching difference between two common approaches to EEG spectral decomposition. In a new analysis of previously published human EEG data, recorded during bilateral rhythmic visual stimulation, we find the typical SSR gain effect when emphasizing stimulus-locked neural activity and the typical retinotopic alpha suppression when focusing on ongoing rhythms. These opposite but parallel effects suggest that spatial attention may bias the neural processing of dynamic visual stimulation via two complementary neural mechanisms

Prestimulus EEG power predicts conscious awareness but not objective visual performance

Prestimulus oscillatory neural activity has been linked to perceptual outcomes during performance of psychophysical detection and discrimination tasks. Specifically, the power and phase of low frequency oscillations have been found to predict whether an upcoming weak visual target will be detected or not. However, the mechanisms by which baseline oscillatory activity influences perception remain unclear. Recent studies suggest that the frequently reported negative relationship between power and stimulus detection may be explained by changes in detection criterion (i.e., increased target present responses regardless of whether the target was present/absent) driven by the state of neural excitability, rather than changes in visual sensitivity (i.e., more veridical percepts). Here, we recorded EEG while human participants performed a luminance discrimination task on perithreshold stimuli in combination with single-trial ratings of perceptual awareness. Our aim was to investigate whether the power and/or phase of prestimulus oscillatory activity predict discrimination accuracy and/or perceptual awareness on a trial-by-trial basis. Prestimulus power (3–28 Hz) was inversely related to perceptual awareness ratings (i.e., higher ratings in states of low prestimulus power/high excitability) but did not predict discrimination accuracy. In contrast, prestimulus oscillatory phase did not predict awareness ratings or accuracy in any frequency band. These results provide evidence that prestimulus power influences the level of subjective awareness of threshold visual stimuli but does not influence visual sensitivity when a decision has to be made regarding stimulus features. Hence, we find a clear dissociation between the influence of ongoing neural activity on conscious awareness and objective performance

Frequency and power of human alpha oscillations drift systematically with time-on-task

Oscillatory neural activity is a fundamental characteristic of the mammalian brain spanning multiple levels of spatial and temporal scale. Current theories of neural oscillations and analysis techniques employed to investigate their functional significance are based on an often implicit assumption: In the absence of experimental manipulation, the spectral content of any given EEG- or MEG-recorded neural oscillator remains approximately stationary over the course of a typical experimental session (∼1 h), spontaneously fluctuating only around its dominant frequency. Here, we examined this assumption for ongoing neural oscillations in the alpha-band (8–13 Hz). We found that alpha peak frequency systematically decreased over time, while alpha-power increased. Intriguingly, these systematic changes showed partial independence of each other: Statistical source separation (independent component analysis) revealed that while some alpha components displayed concomitant power increases and peak frequency decreases, other components showed either unique power increases or frequency decreases. Interestingly, we also found these components to differ in frequency. Components that showed mixed frequency/power changes oscillated primarily in the lower alpha-band (∼8–10 Hz), while components with unique changes oscillated primarily in the higher alpha-band (∼9–13 Hz). Our findings provide novel clues on the time-varying intrinsic properties of large-scale neural networks as measured by M/EEG, with implications for the analysis and interpretation of studies that aim at identifying functionally relevant oscillatory networks or at driving them through external stimulation