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There is a good deal of information available from both neuropsychology and cognitive psychology to support the contention that visual short term memory is functionally segregable from spatial short term memory, within the context of a working memory approach to cognitive functioning. However, relatively little is understood about the precise functional relationships between these segregated components or about the method in which they operate. One suggestion has been that the spatial system operates sequentially, in line with the idea that its output is mediated by movement of the body, whilst the visual short term memory system operates a more parallel manner, retaining input from the visual array in a more holistic fashion. In the research reported in this dissertation, methods originally used to research short term memory in experimental animals were adapted for use in adult humans. This was done with the aim of firstly clarifying the patterns of segregation of visual - spatial working memory and secondly beginning to understand the functional architecture of those segregated components. A series of experiments were conducted, initially based on Logie and Marchetti's (1991) demonstration of visuo-spatial segregation and later based on developments of the delayed response (DR) short term memory task used in experimental animals. In all of these experiments an interference paradigm was used to investigate segregation, with the assumption that segregation would manifest itself in differential effects of interference. For example, visual interference should impair visual but not spatial memory task performance, and vice versa for spatial interference. The results of these experiments clearly demonstrated segregation or visual and spatial processing. Furthermore they support the idea that spatial memory is a sequential process and visual memory a parallel one. However it was also observed that sequentially and parallelism were not the sole specifications of the two systems: memory for the appearance and location of items was also important.

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Neuroimmune proteins are involved in a wide array of biological functions throughout brain development. Importantly, these molecular mechanisms regulate the activity-dependent sculpting of neural circuits during the critical period. Abnormal expression of these molecular mechanisms, especially in early development, is linked to the emergence of neurodevelopmental disorders. Despite having central roles in both normal and pathological conditions, very little is known about the lifespan expression of neuroimmune proteins in the human cortex. As studies exploring the relationship between inflammation and disease tend to rely on animal models, unpacking immune lifespan trajectories in the human brain will be essential for translational research. Furthermore, it will aid the development of timely and effective therapeutic interventions for neurodevelopmental disorders. In my thesis, I characterize the development of 72 neuroimmune proteins in 30 postmortem tissue samples of the human primary visual cortex. These samples cover the lifespan from 20 days to 79 years. I compare the developmental profiles of these immune markers to those of well-studied classic neural proteins including glutamatergic, GABAergic and other synaptic plasticity-related markers. Using a data-driven approach, I found that the 72 neuroimmune proteins share approximately eight developmental patterns, most of which undulate across the lifespan. Furthermore, I used unsupervised hierarchical clustering to show that the development of neuroimmune proteins in the human visual cortex varies from that of classic neural proteins. These findings facilitate a deeper understanding of human cortical development through two classes of proteins involved in brain development and plasticity. / Thesis / Master of Science (MSc) / The human brain develops across the lifespan. This ability of the brain to change and adapt to the environment is called plasticity and it is essential for normal brain functions, such as processing visual information. Immune proteins play important roles in the visual cortex- the brain region responsible for visual information processing. They help establish brain circuits in early development and regulate ongoing neural processes important to brain plasticity. In my thesis, I measure the expression of neuroimmune proteins to unpack their developmental patterns in the human visual cortex. I found that these proteins have fluctuating levels across development, with many displaying heightened expression levels in early childhood. Additionally, I found eight common trajectory patterns that were shared between the proteins. These findings enable a better understanding of how regulators of human brain development mature.

All neuronal circuits of the human brain make up the so-called connectome. Within, three spatial dimensions have been identified, ranging from the microscopic to the mesoscopic to the macroscopic scale. Most studies on connectivity focus on the macroscopic scale by performing magnetic resonance imaging (MRI) in and ex vivo. The bottleneck of this method is the limited resolution accompanied by limited accuracy and validity of 3D MRI data. Thereby, the challenge arises to provide anatomical ground-truth information and create microstructure-informed MRI measurements. One way to overcome these resolution differences between macroscopic and microscopic imaging methods is to generate (sub)cellularly resolved (< 100 nm), mesoscopic tissue blocks (mm-sized). This can be reached, by pairing novel tissue clearing techniques with high-end, large-scale microscopic imaging and is the first goal of the presented thesis. In combining different processing and imaging techniques, that are sensitive to different spatial scales and microstructural properties, a comprehensive understanding of the wiring of the human brain can be generated. Transparency of biological tissue is reached by matching heterogeneous refractive indices (RI) of intra- and extracellular compartments, divided by lipid membranes, to each other and to the RI of their surrounding medium. In doing so, scattering and absorption of light traveling through the sample is reduced. Tissue clearing methods use different approaches to homogenize RI. While hydrophilic and hydrogel-based methods wash out lipids by using strong detergents (e.g., sodium dodecyl sulfate or urea), hydrophobic methods use organic solvents (dibenzylether or ethylcinnamate) to dissolve tissue lipids. The clearing of human brain tissue is particularly challenging as it is densely packed, highly myelinated, and usually aged (i.e., by donors of high age). Initially seven tissue clearing techniques were tested to determine the most efficient one. Efficiency was defined in a high degree of tissue transparency, preserved ability for immunohistochemical staining before or after clearing to obtain highly resolved microscopic imaging results, and short experimental processes. All methods were tested on mm-sized, fixed post mortem brain tissue samples and included the CLARITY, CUBIC, iDISCO, MASH, ECi, Visikol and Ce3D protocol. The CLARITY, CUBIC, iDISCO, and MASH techniques were able to clear aged human brain tissue, whereas the ECi, Visikol and Ce3D techniques were not. Generally, water-based CLARITY and CUBIC methods are gentler than solvent-based iDISCO and MASH techniques. The expansion of CLARITY-treated samples appears to be advantageous as well. However, all technical aspects (i.e., hydrogel pre-treatment, electrophoretic chamber acquisition) of the CLARITY method are time and cost consuming. Here, the iDISCO, MASH, and ECi protocols are more efficient. Their material costs are lower and, if successful, the processing duration is short compared to the CLARITY technique. Although, the Visikol (commercial clearing kit) and Ce3D technique claim to be quickly applied and to clear tissue fast, they are less affordable and more complex to perform as well. After attaining transparency, optical properties of the tissue samples are altered. Hence, the application of immunohistochemistry remains crucial. Microscopic imaging of cleared, immunohistochemically labelled human brain tissue samples revealed that the CLARITY and iDISCO techniques are most suitable. Here, immunohistochemical reagents as well as light penetrated sufficiently deep into the tissue. In addition to the evaluation of different clearing techniques, three microscopic setups, specifically built or equipped for imaging large-scale specimen, were tested to identify suitability, benefits, and downfalls. The results showed that the most suitable and efficient approaches are the combination of i) the CLARITY method coupled with imaging at the Zeiss LSM 880 Airyscan and ii) the iDISCO method coupled with the Miltenyi Biotec Ultramicroscope ll. Both combinations enable three-dimensional histology of aged human brain tissue. There is growing demand for visualizing and examining human cyto- and myeloarchitecture in 3D. Successfully clearing aged human brain tissue whilst preserving its microstructure and ability for immunohistochemical staining will bridge standard histological thin-sectioning (2D) and non-invasive imaging techniques, such as MRI. Once mastered in post mortem human brain blocks in 3D, tools such as fiber tracking and co-localisation are going to provide a powerful tool for multi-modal validation of in vivo microstructure-informed MRI, unraveling the human connectome. Next to unraveling the human connectome based on myelinated axonal fiber pathways, the second goal of this thesis is to investigate the distribution patterns of astrocytes. As they support oligodendrocytes during myelination processes, astrocytes are proposed to be co-localised with myelinated fibers. Revealing their spatial organization offers another great approach to study the wiring of the human brain. Astrocytes are the most common subtype of glia cells in the central nervous system and can serve many functions depending on their interaction partner and surroundings. Those include but are not limited to the regulation of synaptic plasticity, ion and pH homeostasis of neurons and the vasculature. Moreover, astrocytes can adapt morphologically, physiologically, and molecularly during their response to neurodegeneration or demyelination. This remodelling is facilitated, amongst others, by the glial fibrillary acidic protein (GFAP), a cytoskeletal protein and the primary intermediate filament of mature astrocytes. Hence, antibodies against GFAP are commonly and validly used. Furthermore, great heterogeneity occurs among astrocytes with distinguishable morphologies in white matter (fibrous astrocytes) and grey matter areas (protoplasmic astrocytes). Accordingly, GFAP is expressed differently depending on the brain region. Focusing on the co-localisation of myelinated fibers and astrocytes, immunohistochemical analyses were performed to identify myelin- and GFAP-positive structures in aged human cerebral and brainstem areas. Up to three different regions of interest of four different human brains were examined to analyse myelinated fiber and astrocyte content in white and grey matter areas. Subsequently, two image processing approaches were deployed to extract fluorescence intensity values. The first, semi-manual approach, performed with the Zeiss ZEN blue software, generated distribution patterns within a wider range. Hereby, each channel underwent linear unmixing to co-localize the detected fluorescent signals to each other. The second, semi-automated approach, performed with the Segmensation application software, generated distribution patterns within a narrower range. As GFAP dominates in fibrillary astrocytes of white matter regions consistent results were obtained with both image analysis tools. As GFAP expression in grey matter astrocytes is limited, less consistent results were obtained. Therefore, immunohistochemical analyses of different white and grey matter regions of the human brain align with regional distribution patterns of GFAP-positive astrocytes. Remarkably, as proposed in white matter regions, astrocytes and myelinated axons share a similar spatial organization across all investigated regions of interest. In 1992, Suzuki and Raisman first observed this intertwined organization of axons and glial processes in the rat’s brain, coining the term glial framework. The presented thesis confirms this spatial organization of myelinated fibers and astrocytic branches in the human brain. Although this phenomenon was known as well, it was unclear whether this is a global feature. Moreover, the scarcity of studies on the human glial framework, especially in brainstem areas, needs to be acknowledged. This thesis starts to fill the gap, providing evidence for the presence of the glial framework, created by GFAP-positive astrocytes, within white matter regions of cerebral and brainstem areas of the aged human brain. Additionally, the application of immunohistochemistry in cleared, mesoscopic-sized brain tissue samples adds another promising perspective to successfully unraveling the human glial framework globally. As there is growing demand for sensitizing non-invasive imaging techniques to the heterogeneity of astrocytes, studying the spatial organization of myelinated fibers in correlation with astrocytes will provide deeper insights into the impact astrocytes have on neurological integrity and degeneration. Both points of focus of the presented thesis aim to untangle and give a clearer understanding of the human connectome. Myelin, as the primary component of structural and functional integrity of all neuronal fiber pathways, needs to remain the centre of investigations. However, expanding these investigations by studying the glial framework is going to propel our understanding of the human myeloarchitecture forward. Using the coherent approach as is tissue clearing, is a great way to examine structural and functional correlations in 3D and needs to remain a central element of future studies.

Genome wide association studies have provided evidence for a significant association between ZNF804A (zinc finger protein 804A) - specifically the intronic single nucleotide polymorphism (SNP) rs1344706 - and schizophrenia, but little is known about the function of the gene or the effects of the SNP. By studying post-mortem human brain tissue, I characterised ZNF804A immunoreactivity in adult and foetal human brain and investigated effects of diagnosis and rs1344706 genotype on ZNF804A mRNA and protein expression. Secondly, I looked in a large sample of healthy volunteers (n=922) at the effects of rs1344706 on brain structure using volumetry and voxel based morphometry (VBM). Furthermore, I recruited healthy volunteers who were either homozygous for the risk allele or homozygous for the non-risk allele (n=50). They participated in magnetoencephalography (MEG) and magnetic resonance (MR) sessions in which brain activity was measured during a working memory task, a visual processing task, and rest. Using magnetic resonance spectroscopy, also neurotransmitter levels were assessed. The experiments conducted for this thesis showed for the first time that ZNF804A immunoreactivity can be detected in both foetal and adult human brain and that it is mainly localised to layer III pyramidal cells, with a granular subcellular distribution throughout the cytoplasm. No effect of rs1344706 on mRNA and protein expression was found. In our structural MRI study, rs1344706 did not affect macroscopic brain structure as measured by volumetry and VBM, and given the large sample size, this seems a convincing negative. However, we did find that rs1344706 alters prefrontal-hippocampal connectivity, with increased connectivity being observed in risk homozygotes. Additionally, using MEG, we found an effect of ZNF804A genotype on hippocampal connectivity in the theta band (4-8Hz), with non-risk homozygotes displaying more connectivity. This finding provides a first clue as to the mechanisms that might underlie the previously observed effects of rs1344706 on prefrontal-hippocampal connectivity. Future studies will need to elucidate the actual function of the ZNF804A protein, in order to bridge the gap between the molecular and neuroimaging findings described in this thesis.

Neuropeptides are special in their expression profiles restricted to neuronal subpopulations and low tissue mRNA levels. Genetic, epigenetic and transcriptional mechanisms that define spatiotemporal expression of the neuropeptide genes have utmost importance for the formation and functions of neural circuits in normal and pathological human brain. This thesis focuses on regulation of transcription of the opioid/nociceptin genes, the largest neuropeptide family, and on identification of adaptive changes in these mechanisms associated with alcoholism as model human pathology. Two epigenetic mechanisms, the common for most cells in the dorsolateral prefrontal cortex (dlPFC) and the neuron-subpopulation specific that may orchestrate prodynorphin (PDYN) transcription in the human dlPFC have been uncovered. The first, repressive mechanism may operate through control of DNA methylation/demethylation in a short, nucleosome size promoter CpG island (CGI). The second mechanism may involve USF2, the sequence–specific methylation–sensitive transcription factor which interaction with its target element in the CpG island results in USF2 and PDYN co-expression in the same neurons. The short PDYN promoter CGI may function as a chromatin element that integrates cellular and environmental signals through changes in methylation and transcription factor binding. Alterations in USF2–dependent PDYN transcription are affected by the promoter SNP (rs1997794: T&gt;C) under transition to pathological state, i.e. in the alcoholic brain. This and two other PDYN SNPs that are most significantly associated with alcoholism represent CpG-SNPs, which are differentially methylated in the human dlPFC. The T, low risk allele of the promoter SNP forms a noncanonical AP-1–binding element. JUND and FOSB proteins, which may form homo- or heterodimers have been identified as dominant constituents of AP-1 complex. The C, non-risk variant of the PDYN 3′ UTR SNP (rs2235749 SNP: C&gt;T) demonstrated significantly higher methylation in alcoholics compared to controls. PDYN mRNA and dynorphin levels significantly and positively correlated with methylation of the PDYN 3′ UTR CpG-SNP suggesting its involvement in PDYN regulation. A DNA–binding factor with differential binding affinity for the T allele and methylated and unmethylated C alleles of the PDYN 3′ UTR SNP (the T allele specific binding factor, Ta-BF) has been discovered, which may function as a regulator of PDYN transcription. These findings emphasize the complexity of PDYN regulation that determines its expression in specific neuronal subpopulations and suggest previously unknown integration of epigenetic, transcriptional and genetic mechanisms that orchestrate alcohol–induced molecular adaptations in the human brain. Given the important role of PDYN in addictive behavior, the findings provide a new insight into fundamental molecular mechanisms of human brain disorder. In addition to PDYN in the dlPFC, the PNOC gene in the hippocampus and OPRL1 gene in central amygdala that were downregulated in alcoholics may contribute to impairment of cognitive control over alcohol seeking and taking behaviour.

The effects of mobile phone-like electromagnetic radiation on the human brain activity are examined. The research focuses on both radio frequency (RF) exposures and the much less studied low frequency (ELF) exposures (less than 40 kHz) arising from the battery operation of GSM handsets. The first single blind study recruited a small sample of twelve human volunteers. The eyes closed resting EEG activity is monitored after radio frequency exposure. With SAR levels of 2 W/kg, results reveal no statistical changes in any of the examined frequency bands for neither pulsed modulated RF signals nor continuous wave RF signals. In the second double blind study, a sample of 72 volunteers is recruited and an improved protocol comprised of separate pulsed RF, continuous RF and pulsed ELF exposures is employed. Exposures are delivered through a custom made handset capable of independent RF and ELF exposures. Findings include a reduced alpha band frequency activity during pulsed radio frequency and low frequency radiations exposures but no changes under the continuous RF radiation. Changes are present both during as well as after exposure, while greater changes are observed during exposures. The study of some non linear measures of the resting EEG revealed no changes under any of the active exposures. As the observed changes are very close to the normal EEG variation during resting conditions, their biological significance and health impact is not immediately obvious. However, their mere demonstration points to a low level interaction mechanism which may deserve further study.