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Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Autism is a severe childhood neuropsychiatric condition characterized by impairments in socialization and communication, and by restricted and repetitive behaviours. Autism spectrum disorder (ASD) is a complex and largely unknown disease with a strong genetic basis, multiple genes involved and environmental factors determining its phenotype. Interestingly, the DLX1/DLX2 and DLX5/DLX6 bigene clusters are located in autism susceptibility loci and Dlx genes are involved in GABAergic interneurons differentiation and migration to the cortex during forebrain development. Dlx gene expression is controlled by different cis-regulatory elements. Of these, 4 are active in the forebrain, URE2, I12b, I56ii and I56i. In order to determine the role of the DLX genes in ASD, variants were found in gene exons and in cis-regulatory elements in autistic individuals. A single nucleotide polymorphism (SNP), a change of an adenine for a guanine, was identified in I56i enhancer. Finding a SNP in I56i was very surprising considering that it is located in a Dlx binding motif highly conserved among >40 species. We showed, using in vitro approaches, that the presence of this SNP affects the affinity of Dlx for their binding site and reduces the transcriptional activation of the enhancer. The SNP also affects activity of the I56i enhancer in transgenic mice. In order to determine the real impact of the SNP in vivo, mutant mice harboring the SNP in their I56i enhancer were produced. That involved the insertion of the I56i enhancer with the SNP, using homologous recombination in mouse embryonic stem cells to replace the wild type version of the enhancer. With these mutant mice, we demonstrated that, in vivo, this SNP reduces Dlx5 and Dlx6 expression in the forebrain. Furthermore, this decrease in Dlx5/Dlx6 expression could affect the differentiation and/or migration of specific populations of inhibitory interneurons in the forebrain. No distinct iv behavioural phenotypes were observed between wild type mice and those carrying the SNP, during social interaction and anxiety tests. Therefore, these results suggest that even a subtle change in a regulatory element can have an impact in the development of the forebrain and may even contribute to disorders such as autism.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disease characterized by progressive muscle-wasting and loss of mobility. One-third of patients with DMD are also affected by cognitive impairments such as a lower than average IQ and impaired working memory, comorbid with neuropsychiatric disorders such as anxiety and autism-related behaviours. DMD is caused by mutations in the DMD gene resulting in the deletion of the full-length dystrophin protein (Dp427) and, dependent on mutation, other dystrophin isoforms. These isoforms are predominantly found in the brain and deletion may impact on cognition. The most commonly used animal model to study DMD is the mdx mouse which completely lacks Dp427 but no other DMD isoforms. Although the muscle phenotype is well-established, behavioural characterization of the mdx mouse model has been inconclusive. In this thesis I investigated the hippocampal and amygdala cellular and behavioural phenotypes of the mdx mouse. I show that post-natal neural stem-like cell division in the SGZ is altered in the absence of Dp427 resulting in enhanced symmetric division. I show in vitro that primary mdx cultures are fewer and smaller than wild-type, consistent with an increase in symmetrical self-renewal whereas secondary cultures are fewer and larger, consistent with a shift in symmetric division producing transit-amplifying type 2a daughter cells. I next characterized the mdx mouse model using a battery of behavioural tests. Data presented here show that mdx mice do not exhibit an anxious phenotype, do not display autism-related behaviours, and do not display impairments in and spatial learning or memory. However, associative learning, as measured in the fear conditioning paradigm is enhanced in mdx mice. Lastly, I attempted to generate three different brain-specific dystrophin knock-out mouse models to examine role of other dystrophin isoforms. While none of the models were able to deplete dystrophin from brain, given the inverse relationship between Cre-mediated efficiency and the genetic distance of the loxP sites in the fDMDH mouse employed, I do provide important insight into the presence and absence of the muscle-specific enhancers in constructs commonly used to generate brain-specific mouse models. Taken together, this thesis provides converging evidence to indicate that loss of Dp427 impacts on fear associative learning and stem-cell like division in the SGZ but likely does not underlie the non-progressive cognitive impairments affecting one-third of all DMD patients.

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Die Erforschung der Zusammenhänge zwischen Gehirnplastizität und individuellem Verhalten gestaltet sich aufgrund ihrer Komplexität im Tiermodell schwierig. Die vorliegende Studie wurde im mit dem Ziel konzipiert, die Individualitätsentwicklung bei Mäusen mit den gleichen physiologischen und genetischen Voraussetzungen in einer komplexen räumlichen und sozialen Umgebung zu beschreiben. Ich untersuchte die Korrelation dieser Entwicklung mit der Neurogenese im adulten Hippokampus als Maß für Gehirnplastizität. Zu diesem Zweck wurden zwei je mit einem automatisierten RFID-Tracking-System ausgestattete Großgehege mit jeweils 40 Tieren besiedelt. Die Bewegungen der Tiere wurden kontinuierlich aufgezeichnet und es wurden zudem direkte Verhaltensbeobachtungen durchgeführt. Die Tiere zeigten eine normale physiologische Entwicklung. Die Roaming Entropy (RE), ein Maß für die Gleichmäßigkeit, mit der die Tiere ihr Gehege nutzten, beschreibt das Erkundungsverhalten der einzelnen Mäuse. Die kumulativ erworbenen RE-Werte (cRE) in jedem der beiden Gehege wurden mit der Zeit zunehmend verschieden. Es war nicht möglich, aufgrund kleiner anfänglicher Unterschiede die Endwerte zu berechnen. Das bedeutet, dass die Tiere erst durch die andauernde Interaktion mit ihrer Umwelt und den Artgenossen unterschiedlicher wurden. Darüber hinaus sind die cRE-Werte am Endpunkt positiv mit den Neurogenesewerten korreliert. Dies beweist, dass während der Entwicklung auftretende Faktoren die Individualitätsentwicklung beeinflussen. Dieser Prozess benötigt plastische Hirnstrukturen und formt diese wiederum. Die Verhaltensanalysen zeigten, dass Tiere, die viele Antennenkontakte sammelten („most active“, MA) nicht zwangsläufig auch hohe cRE-Werte hatten. MA-Mäuse waren häufiger an sozialen Interaktionen beteiligt als Tiere mit wenigen Antennenkontakten („least active“, LA), akkumulierten über die Zeit niedrigere cRE-Werte und standen vermutlich weiter unten in der sozialen Hierarchie. Zusammenfassend kann man sagen, dass das Ausmaß der räumlichen Exploration und die allmähliche Erweiterung der Erfahrung mit einer gesteigerten Plastizität des Gehirns in Form von adulter Neurogenese verbunden war. Die Daten zeigen, dass Tiere mit den gleichen Voraussetzungen sich dennoch auf zunehmend divergierende, individuelle Art entwickeln. Dies ist zumindest teilweise durch leicht unterschiedliche epigenetische Voraussetzungen zu erklären, die durch das Wechselspiel mit dem komplexen Umfeld weiter auseinanderdriften. Auch scheint es, dass Individuation lebenden Organismen inhärent und Voraussetzung für evolutionäre Prozesse ist. Die Studie zeigt, dass die Unterschiede in individuellem Verhalten und Gehirnstruktur nicht allein durch Genen und Umweltbedingungen festgelegt sind, sondern auch durch Faktoren, die sich während der ontogenetischen Entwicklung entfalten, beeinflusst werden. Der beschriebene Versuchsaufbau stellt darüber hinaus ein Tiermodell für die Untersuchung von Mechanismen und modulierenden Faktoren auf die strukturellen Grundlagen der Plastizität als individuelle Reaktion auf die gemeinsam genutzte Umgebung dar. / The complex nature of the relationship between brain plasticity and individual behaviour renders its investigation using animal models difficult. The present study was designed to describe the emergence of individuality in mice with the same physiological, environmental and genetic preconditions in response to complex environmental and social cues. I investigated the correlation of this development to brain plasticity, namely neurogenesis in the adult hippocampus. To this end, two large, multi-level enclosures fitted with and automated RFID tracking system were populated with 40 animals to each. The mice were continuously tracked and live behaviour observations were done. The animals showed normal physiological development. The Roaming Entropy (RE), a measure for the evenness of their usage of the enclosure, describes the exploration behaviour of each animal. Cumulatively acquired RE scores (cRE) within an enclosure increasingly diverged with time. Small differences at the beginning were not predictive of the end values. Thus, the animals became different through the continued interaction with environment and conspecifics. Moreover, the cRE values at the end point positively correlated with the amount of hippocampal neurogenesis. This proves that factors emerging during development contribute to individuality development. These factors at the same time shape and rely on plastic brain structures. Behavioural analyses showed that animals with a high amount of antenna contacts (most active, MA mice) were not necessarily those with high cRE values. MA mice were more often involved in social interactions than the least active mice (least active, LA), accumulated lower cRE scores over time and seemed to be lower in the social hierarchy. In conclusion, the amount of spatial exploration and gradual broadening of experience was linked to brain plasticity in the form of elevated levels of hippocampal neurogenesis. The data shows that animals with same preconditions still develop along increasingly divergent, individual paths. This is probably partly given through slightly different epigenetic preconditions, drifting further apart by interaction with the complex environment. Also, individuation seems to be inherent in living organisms and necessary for evolutionary processes. The study shows firstly that differences in individual behaviour and brain structure are defined not only by genes and the environment but also modulated by factors unfolding or emerging during ontogenetic development. The present paradigm moreover introduces an animal model for studying mechanisms and influences on the structural basis of plasticity as an individual response to the nonshared environment.

This thesis analyses how users' mouse behaviour during a video stall gets influenced by the type of video, either branched or linear, and by the presence of a playback bar. An experiment was conducted with thirty-two participants divided into six groups. Each group was watching a different combination of four videos with stalls, the first two videos belonging to the same type of video and either with or without a playback bar, and the last two videos changed in one of the two aspects. With mouse-tracking, these aspects were studied through the variables of mouse activity, average speed, average distance between the cursor and the playback bar, and the total distance moved on the screen. The participants also filled in questionnaires about their mouse behaviour, after watching each video, and their answers were later analysed through a thematic analysis. The results showed no significant differences between the groups in any of the main dependent variables. In general, within all groups, the participants moved the mouse very scarcely, indicating that the results about mouse movement should be interpreted carefully. During videos with a playback bar, mouse movements appeared to be concentrated to the stalls, focusing the movements to the bottom of the screen where the playback bar is located. Mouse behaviour during videos without a playback bar was more evenly divided between the different parts of the video and of the screen, or the user were not moving the mouse at all. Watching branched or linear videos influenced the mouse behaviour in such a way that branched videos seemed to engage the user to interact with the video player more than the linear videos. However, no difference was noticed between these conditions for active users during stalls when a playback bar was present. The thematic analysis gave clear indications that the playback bar was an important component for understanding a stall. Based on these findings, conclusions are drawn that a stall is a situation of watching videos during which mouse behaviour may be less affected by the type of video, and more influenced by the access to a playback bar. The playback bar was shown to be a source of information about the system and the situation.