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A current challenge in cognitive neuroscience is to provide an explicit separation of the neural correlates of abstract global decision variables from sensory and integrative ones. In particular, the insular cortex and the adjacent frontal operculum seem to have a crucial but still unclear role in evidence accumulation and decision signaling in perceptual decision-making tasks. Here, we have used a visual decision-making paradigm based on the detection of ambiguous two-tone (Mooney) face stimuli to assess the emergence of holistic percepts. These are constructed using global gestalt rules and not by gradual spatiotemporal increases in sensory evidence. Our paradigm (neurochronometric approach) enabled the experimental separation between multiple cognitive components in perceptual decision validated by both model-driven and data-driven analysis. This strategy allowed for the functional dissection of operculo-insular networks into task related complexes such as anterior (accumulator), middle (decision) and posterior (somatosensory/sensorimotor). We conclude that global perceptual integration based on holistic rules requires a distributed operculo-insular network.

The use of group-housed rodents in many fields of biomedical research imposes a need to identify individuals in a cage. Few studies have been designed to assess possible negative effects of identification methods of newborn mice on their development and wellbeing. In the present study, three different identification methods were applied to newborn C57BL/6J mice on postnatal day (pnd) 5 (toe clipping, toe tattoo ink puncture and subcutaneous implantation of a small transponder). All identification methods used proved to be effective for long-term marking of individual animals. Newborn mice showed the least reaction to toe clipping followed by toe tattoo ink puncture and transponder implantation was the most distressful individual identification procedure in newborn mice. Importantly, clipped toe tissue proved to be enough for genotyping purposes. No overall consistent differences in somatic and neurological reflex development during the postnatal period were shown as a result of the newborn individual identification procedures used. Further, none of the methods interfered significantly with the adult animals' general normal behaviour (e.g. ability to move, grasp, climb) and sensory-motor functions as assessed with a simplified SHIRPA battery of tests, as well as Rotarod and Elevated Plus Maze tests. Postmortem thymus and adrenal gland weights gave no indication of chronic stress as a consequence of the identification method. We conclude that toe clipping might even be advisable in newborn mice at a very young age, when genotyping is needed. Toe tattoo ink puncture is also a good identification method for newborn mice and transponder implantation should only be used in older newborns or applied at weaning., As this study was performed within the framework of the first author’s master thesis, thanks are due to Laboratory Animals Ltd for providing financial support.

The relation of gamma-band synchrony to holistic perception in which concerns the effects of sensory processing, high level perceptual gestalt formation, motor planning and response is still controversial. To provide a more direct link to emergent perceptual states we have used holistic EEG/ERP paradigms where the moment of perceptual “discovery” of a global pattern was variable. Using a rapid visual presentation of short-lived Mooney objects we found an increase of gamma-band activity locked to perceptual events. Additional experiments using dynamic Mooney stimuli showed that gamma activity increases well before the report of an emergent holistic percept. To confirm these findings in a data driven manner we have further used a support vector machine classification approach to distinguish between perceptual vs. non perceptual states, based on time-frequency features. Sensitivity, specificity and accuracy were all above 95%. Modulations in the 30–75 Hz range were larger for perception states. Interestingly, phase synchrony was larger for perception states for high frequency bands. By focusing on global gestalt mechanisms instead of local processing we conclude that gamma-band activity and synchrony provide a signature of holistic perceptual states of variable onset, which are separable from sensory and motor processing.

Huntington's disease (HD) is a neurodegenerative disorder causing cognitive and motor impairments, evolving to death within 15-20 years after symptom onset. We previously established a mouse model with the entire human HD gene containing 128 CAG repeats (YAC128) which accurately recapitulates the natural history of the human disease. Defined time points in this natural history enable the understanding of longitudinal trajectories from the neurochemical and structural points of view using non-invasive high-resolution multi-modal imaging. Accordingly, we designed a longitudinal structural imaging (MRI and DTI) and spectroscopy (1H-MRS) study in YAC128, at 3, 6, 9 and 12 months of age, at 9.4 T. Structural analysis (MRI/DTI), confirmed that the striatum is the earliest affected brain region, but other regions were also identified through connectivity analysis (pre-frontal cortex, hippocampus, globus pallidus and thalamus), suggesting a striking homology with the human disease. Importantly, we found for the first time, a negative correlation between striatal and hippocampal changes only in YAC128. In fact, the striatum showed accelerated volumetric decay in HD, as opposed to the hippocampus. Neurochemical analysis of the HD striatum suggested early neurometabolic alterations in neurotransmission and metabolism, with a significant increase in striatal GABA levels, and specifically anticorrelated levels of N-acetyl aspartate and taurine, suggesting that the later is homeostatically adjusted for neuroprotection, as neural loss, indicated by the former, is progressing. These results provide novel insights into the natural history of HD and prove a valuable role for longitudinal multi-modal panels of structural and metabolite/neurotransmission in the YAC128 model.

Blood-brain barrier (BBB) disruption is a common feature in neurodegenerative diseases. However, BBB integrity has not been assessed in spinocerebellar ataxias (SCAs) such as Machado-Joseph disease/SCA type 3 (MJD/SCA3), a genetic disorder, triggered by polyglutamine-expanded ataxin-3. To investigate that, BBB integrity was evaluated in a transgenic mouse model of MJD and in human post-mortem brain tissues.Firstly, we investigated the BBB permeability in MJD mice by: i) assessing the extravasation of the Evans blue (EB) dye and blood-borne proteins (e.g fibrinogen) in the cerebellum by immunofluorescence, and ii) in vivo Dynamic Contrast Enhanced-Magnetic Resonance Imaging (DCE-MRI). The presence of ataxin-3 aggregates in brain blood vessels and the levels of tight junction (TJ)-associated proteins were also explored by immunofluorescence and western blotting. Human brain samples were used to confirm BBB permeability by evaluating fibrinogen extravasation, co-localization of ataxin-3 aggregates with brain blood vessels and neuroinflammation.In the cerebellum of the mouse model of MJD, there was a 5-fold increase in EB accumulation when compared to age-matched controls. Moreover, vascular permeability displayed a 13-fold increase demonstrated by DCE-MRI. These results were validated by the 2-fold increase in fibrinogen extravasation in transgenic animals comparing to controls. Interestingly, mutant ataxin-3 aggregates were detected in cerebellar blood vessels of transgenic mice, accompanied by alterations of TJ-associated proteins in cerebellar endothelial cells, namely a 29% decrease in claudin-5 oligomers and a 10-fold increase in an occludin cleavage fragment. These results were validated in post-mortem brain samples from MJD patients as we detected fibrinogen extravasation across BBB, the presence of ataxin-3 aggregates in blood vessels and associated microgliosis.Altogether, our results prove BBB impairment in MJD/SCA3. These findings contribute for a better understanding of the disease mechanisms and opens the opportunity to treat MJD with medicinal products that in normal conditions would not cross the BBB.

Made available in DSpace on 2021-12-23T14:16:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-29 The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), Minho University, and University of Beira Interior. Financial support was provided by grants from the Tecniplast S.p.A.and Ultragene Lda; by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020; and by the projects NORTE-01-0145- FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).