Visuomotor integration and visuomotor skill learning depend on local plasticity in visual cortex during development

Visuomotor experience shapes responses in visual cortex during development. Coupling between movement and visual feedback establishes a comparator circuit between top-down and bottom-up inputs in layer 2/3 of mouse primary visual cortex (V1). Such a circuit is capable of computing prediction error responses in layer 2/3 excitatory neurons in V1. Given that visual cortex receives both the bottom-up visual input and signals consistent with a top-down prediction of visual flow given movement, it has been speculated that visual cortex is a site of integration of these two signals. If correct, we would predict that perturbing plasticity in V1 during development should prevent the establishment of a normal balance between bottom-up and top-down input, and consequently an impairment of visuomotor prediction errors in layer 2/3 neurons of primary visual cortex. In Chapter I, we tested whether local plasticity in visual cortex is necessary for the establishment of this balance by locally perturbing neural plasticity. Our results show that perturbing NMDA receptor-dependent plasticity during development of the visual system leads to a reduction in visuomotor prediction error responses, and that plasticity in V1 is crucial for the development of normal visuomotor integration. In Chapter II, we further investigated the balance of top-down and bottom-up inputs in V1 and ask, given that pro-psychotic agents (e.g., hallucinogens) can influence visual cortex activity, whether antipsychotic drugs also induce common circuit changes. We investigated three antipsychotic drugs: Haloperidol, Clozapine and Aripiprazole, with the aim of identifying a common functional signature, possibly underpinning their clinical efficacy. The most common change was a decrease in visuomotor prediction errors in layer 2/3 neurons. Clozapine, as one of most effective drugs, decreased activity of inhibitory neurons thought to mediate visual feedforward signals and increased the mean activity in layer 5. Overall, however, we did not find common changes in all of these three antipsychotic drugs

Neonatal Stimulation of PKC Epsilon Signaling Normalizes Fragile X-Associated Deficits in PVN Oxytocin Expression and Later-Life Social and Anxiety Behavior

Fragile X Syndrome (FXS) is an inherited developmental disorder characterized by disturbances in emotional and social behavior. Our studies have revealed suppressed hippocampal PKCε expression in Fmr1 knockout (KO) mice, the leading model of FXS. To compensate for this deficiency, we stimulated PKCε in neonatal KO mice by administering a selective PKCε activator, dicyclopropyl-linoleic acid (DCP-LA), and studied its effect on ventral hippocampal neurons and a proximal target of the ventral hippocampus, the hypothalamus, which regulates social and emotional behavior. We observed that at postnatal day 18 (P18), vehicle-treated KO mice displayed increased surface localization of the 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR2 in the ventral CA1 region, indicative of increased neuronal excitability. Since the hippocampus is known to exert an inhibitory influence on the hypothalamus, we tested if this possible CA1 stimulation was associated with a suppression of oxytocin synthesis in the hypothalamus. Intriguingly, the number of oxytocin+ cells in the hypothalamic paraventricular nucleus (PVN) of P20 KO mice was sharply suppressed. However, both the increased surface localization of GluR2 and the suppression of PVN oxytocin+ cells in the KO mice were rescued by DCP-LA treatment from P6-14, to levels comparable to that in the wild-type controls. Moreover, this neonatal treatment regimen was able to fully rescue hyper-anxiety and social behavior deficits in adult (\u3eP60) KO mice. Thus, we present a novel strategy to circumvent aberrant brain development in FXS and accompanying behavioral deficits, by activating PKCε signaling during neonatal development

Investigating the Effects of Chronic Amphetamine Treatment on the Midbrain Superior Colliculus

Heightened distractibility, the reduced ability to filter out irrelevant information, is a disruptive symptom found in numerous conditions and healthy ageing. It is most effectively treated with psychostimulant drugs, such as amphetamine, which are given chronically in attention deficit hyperactivity disorder (ADHD). There is converging evidence linking the superior colliculus (SC) to the regulation of distractibility, and acute amphetamine administration is known to suppress collicular responsiveness, however relatively little is known about its mechanism of action and long lasting effects when administered chronically. It is therefore the aim of this thesis to investigate the effects of chronic treatment with amphetamine on the SC, as a neural correlate of distractibility. To achieve this, adolescent Hooded Lister rats were treated orally with amphetamine (2, 5 or 10 mg/kg) or a control (vehicle or untreated) for one month. The effects of treatment were then explored by investigating: collicular-dependant and locomotor behaviour; visual responsiveness of the superficial SC; responses to an acute amphetamine challenge; and the morphology of the superficial SC. At high doses, oral amphetamine treatment resulted in the development of locomotor tolerance, in contrast to previous research using i.p. administration which report sensitisation. Evidence of suppression of activity in the SC was identified from a reduced ability to perform collicular dependant behaviours, and from weaker light responsiveness measured from multi-unit activity in the superficial SC following treatment with high doses of amphetamine. Evidence was also found of a potential compensatory mechanism involving synaptophysin expression and enhanced peri-synaptic activity. This thesis also investigated the types of dendritic spines prevalent in the superficial SC for the first time, and found that these structures were unaffected by amphetamine treatment. These results indicate that the therapeutic effects of amphetamine may stem from suppression of collicular activity, and the SC is susceptible to amphetamine induced remodelling

Skin cell heterogeneity and dynamics during morphogenesis, tissue homeostasis, and regeneration

Skin is our protective barrier against various environmental harms. For the skin to fulfill its crucial function, it relies on multiple cell types working in concert; but most importantly it relies on skin-resident epithelial stem cells. These cells ensure an intact barrier through constant replacement of the epidermis and they ensure proper hair production through cyclical regeneration of hair follicles. This combination of constant and cyclical renewal within one tissue makes skin a prime model system for the study of adult tissue stem cells. The overall aim of this thesis was to transcriptionally dissect this well-established model system in a systematic and unbiased way. The majority of data presented in this thesis is based on the combination of single-cell RNA sequencing and in situ stainings of mRNA. This combination allows us to appreciate the genome-wide transcriptional heterogeneity while still being able to place the identified cell populations in their spatial tissue context. In Paper I, the first whole-transcriptome study of skin at the single-cell level, we examined the vectors describing cellular heterogeneity within the epidermal compartment of mouse skin during its resting stage (telogen). In Paper II, we expanded on this analysis by including full-thickness skin during rest (telogen) and growth (anagen). This allowed for an unbiased census of all major cell types contained in the skin, and it furthermore enabled us to study how skin achieves and accommodates hair growth. In Paper III, we studied the role of dermal fibroblasts in early embryonic skin development. We uncovered unexpected heterogeneity among embryonic fibroblasts and explored their supportive functions for skin maturation. Moreover, we identified novel keratinocyte subpopulations and closely analyzed epidermal fate decisions. In Paper IV, we monitored transcriptional adaptations of two distinct epidermal stem cell populations during their contribution to wound healing. This allowed us to answer fundamental questions about stem cell plasticity and the dynamics of cell adaptations following injury. In sum, this thesis uncovers the dynamic and heterogeneous nature of mouse skin during adult tissue homeostasis, embryonic development, and tissue regeneration after injury. Most importantly, we provide new insights into how stem cell identity is shaped and how developmental as well as regenerative processes are orchestrated

Microglial clearance function: dependence on phenotypes

Dissertação para obtenção do Grau de Mestre em Genética Molecular e BiomedicinaMicroglia are active sensors of the brain and respond promptly to even minor disturbance in their microenvironment. A feature of this response is the accumulation of these cells at the site of lesion. Neonatal jaundice is a common condition of the newborn and may determine injury to neurons and glial cells, such as microglia, when levels of unconjugated bilirubin (UCB) are excessive. With the objective to evaluate whether microglia have a protective or deleterious role, we decided to assess, using the Boyden chamber, the chemotactic effect of free unbound UCB (fUCB), as well as the migration ability of UCB-treated microglia in the absence or in the presence of chemotatic compounds, such as ATP and S100B. Also, we intended to evaluate the effect of glycoursodeoxycholic acid (GUDCA) as a modulator. To characterize our usual model of microglia isolation, phenotypic evaluation of cultures with different days in vitro (DIV) was performed by estimating cell morphology, nuclear factor-kappaB (NF-κB) activation and phagocytic ability. We observed that fUCB did not act as a chemotactic compound for microglia and that cells treated with UCB showed decreased migration ability. Co-incubation with GUDCA prevented this effect and enhanced microglia migration. However, reduced effects were observed in the presence of ATP and abolished when using S100B. Isolated microglia with 2 DIV showed features of activation, but presentedramified morphology of the “resting” state, less NF-κB activation and increased phagocytosis at 13 DIV. Data indicate that microoglia exposure to UCB leads to a reduced migration ability and that co-incubation with GUDCA prevents this deleterious effect, resulting in an increased migration. Characterization of microglia phenotypes, along the time in culture, point to 13 DIV cells as the most suitable for studies intended to evaluate microglia reactivity to UCB, and probably to other stimuli

Designing a brain-based learning environment

The purpose of this project was to develop a teacher friendly guide that would help teachers not only apply brain-based strategies in the classroom, but also to see results from transforming their classrooms into brain-based learning environments

Affective Regulation Through Touch: Homeostatic and Allostatic Mechanisms

We focus on social touch as a paradigmatic case of a unifying perspective on the embodied, cognitive and metacognitive processes involved in social, affective regulation. Social touch appears to have three interrelated but distinct functions in affective regulation. First, it regulates affects by fulfilling embodied expectations about social proximity and attachment, mostly likely by convergent hedonic, dopaminergic and analgesic, opioidergic pathways. Second, caregiving touch such as feeding or warming an infant regulates affect by socially enacting homeostatic control and co-regulation of physiological states, most likely by corresponding ‘calming’ autonomic and endocrine pathways. Third, affective touch such as gentle stroking, kissing or tickling regulates affect by allostatic regulation of the salience and epistemic gain of particular experiences in given contexts and timescales, possibly regulated by oxytocin release and related ‘salience’ neuromodulators and circuits.</p

Role of glial cells as contributors to the onset and propagation of als disease

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2017Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease comprehending critical neuroinflammatory pathways, where microglia and astrocytes play a crucial role. ALS onset events are largely unknown and identification of disease steps during progression and dissemination, including the possible role of exosomes, are not clarified. Several models were used to improve data validity and deepen knowledge in ALS. We identified innovative targets to regulate microglia M1 polarization, including NLRP3-inflammasome, HMGB1 alarmin and MFG-E8/lactadherin, and demonstrated the sorting of microglial microRNA(miR) 155/miR-146a into exosomes. We showed that ALS NSC-34 MNs and their exosomes are enriched in miR-124, which are captured and drive early N9-microglia M1 polarization, with later development of M1/M2 subpopulations containing increased miR-124/miR-146a/miR-155. Moving from in vitro models to the spinal cord of the SOD1G93A ALS mouse model, we observed that depressed intercellular communication and increased miR-155 were early disease events preceding the inflammatory status of the symptomatic stage. Upregulated CX3CL1-CX3CR1, connexin-43/pannexin-1 and miR-124/miR-125b/miR-146a/miR-21 emerged as candidate targets for pathological neuroinflammation. Reduced MN number, together with aberrant/reactive astrocytes showing deficient glutamate transporters and GFAP, additionally characterized such state. Differently deregulated profiles of microglia isolated from the spinal cord of 7-day old SOD1G93A mice, after short- and long-term cultures, highlighted that cells present transient phenotypes accordingly to ALS environmental progression-stimuli and ultimately acquire a less responsive phenotype to stimulation. Astrocytes isolated from these mice promoted diverse inflammatory polarized subtypes in wild-type and ALS microglia, thus accounting to microglia heterogeneous populations, while strengthened deregulated microglia-astrocyte cross-talk as part of ALS neurodegenerative mechanisms. Our studies in ALS models reveal early promising biomarkers and novel targets to control excessive neuroinflammation and spread, including exosomal microRNAs. Due to multiple microglia phenotypes induced by MNs and their exosomes, and by reactive astrocytes, in the ALS disease, differentiated and combined therapeutic approaches may be recommended.Santa Casa da Misericórdia de Lisboa, programa de Investigação Científica em Esclerose Lateral Amiotrófica, projeto ELA-2015-002, The EU Joint Programme-Neurodegenerative Disease Research (JPND), projeto JPCOFUND/003/201