Neonatal Sprague-Dawley rat pups were assigned to one of five groups. Three cocaine-treated groups were injected SC with either 40, 60, or 80 mg/kg/day of cocaine from postnatal day (PND) 4 through 9. Control groups were either injected with equivalent volumes of sterile dH2O (vehicle control) or received no injections (normal control) from PND 4 through 9. This early postnatal period, corresponding to the third trimester of pregnancy in humans, is characterized as a period of rapid development within the central nervous system (CNS), generally termed the brain growth spurt. The survival rate, somatic growth, and brain development in response to the various dosages of postnatal cocaine administration were assessed. There was a dose-dependent relationship between cocaine administration and survival rate. Furthermore, significantly reduced somatic growth, assessed in terms of body weight, was found in animals given 80 mg/kg cocaine daily, as compared with controls. With respect to brain weight, no significant differences were obtained among the various doses of cocaine-treated and control animals and there was no evidence of regional vulnerability (forebrain, cerebellum, or brainstem) to the cocaine insult. Additionally, neither an effect of gender, nor the interactions of gender with various doses of cocaine treatment was found on somatic growth and brain development. Taken together, the present results suggest that the brain exhibits a greater resistance to the cocaine insults than does somatic growth. Several possible explanations regarding the somatic growth retardation are discussed.

This study was undertaken to investigate whether the developing brain adapts at biochemical level against neonatal hypothyroidism, as it does so against a variety of physiological disturbances. A moderate hypothyroid state in mice neonates was induced by supplementing 0.05% methimazole in drinking water to the mothers up to suckling period, and its effect on concerted development of the enzymes regulating metabolic channeling of glucose vis a vis glucose phosphorylating activity were studied. In the brain of control mice, the activity of glucose-6-phosphate dehydrogenase (G6PDH), that channels glucose in biosynthetic route (Pentose phosphate pathway, PPP), increased slightly (∼ 1.3 times) from day1 to 10w age. However, glucose phosphorylating activity and the enzymes that commit glucose for energy production, viz phosphofructokinase1 (PFK1), pyruvate kinase (PK) and lactate dehydrogenase (LDH) showed a progressive postnatal increase to attain their respective adult levels (∼ 5-10 times higher than 1day value) by 3-10w ages of mice. In comparison to the control, in the brain of age matched neonatal hypothyroid mice, glucose phosphorylating activity, G6PDH and PFK1 increased significantly (p<0.001) at day1. Thereafter, though, glucose phosphorylating activity continued to increase up to 1w age and remained static thereafter, G6PDH declined significantly (p<0.001) from 1w onward ages. On the other hand, as PFK1 activity increased significantly up to 10w age (p<0.001), the ratio of G6PDH/PFK1 showed a marked decline from 1w onward ages. PK and LDH also showed increasing trend up to 3w age in the brain of hypothyroid mice pups. The results suggest that a moderate hypothyroid state, during the period of rapid brain growth (day 1-1w age), stimulates all the enzymes that regulate channeling of glucose in both, the energy yielding and biosynthetic paths. However, the later postnatal ages, it modulates these enzymes in a metabolic path dependent manner.

The prefrontal cortices, in particular lateral prefrontal cortex (LPFC) and anterior cingulate cortex (ACC), have been implicated in top-down control of attention switching and behavioral flexibility. These cortices and their networks are disrupted in autism, a condition in which diverse behaviors such as social communication and attention control are dysregulated. However, little is known about the typical development of these cortical areas or the ways in which this process is altered in neurodevelopmental disorders. In order to identify changes that could affect the local processing of signals transmitted by the short-range pathways connecting the ACC and LPFC I assessed developmental changes in the distinct cortical layers, which send and receive different pathways and have unique inhibitory microenvironments that dictate excitatory-inhibitory balance. Normative developmental trends were compared with those seen in individuals with autism to identify changes that may contribute to symptoms of attention dysfunction. Unbiased quantitative methods were used to study overall neuron density, the density of inhibitory neurons labeled by the calcium-binding proteins calbindin (CB), calretinin (CR), and parvalbumin (PV), and the density, size, and trajectory of myelinated axons in the individual cortical layers in children and adults with and without a diagnosis of autism. There was a reduction in neuron density and an increase in the density of myelinated axons in both areas during neurotypical development. Axons in layers 1-3 of LPFC were disorganized in autism, with increased variability in the trajectory of axons in children and a decrease in the proportion of thin axons in adults. These findings were most significant in layer 1, the ultimate feedback-receiving layer in the cortex. While there were no differences in neuron populations between cohorts in children, in adults with autism there was a significant reduction in the density of CR-expressing neurons in LPFC layers 2-6 and a significant increase in the density of PV-expressing neurons in ACC layers 5-6. In autism, these findings suggest that dysregulation of the normal development of axonal networks, seen in children, may induce compensatory developmental changes in cell and axon populations in adults that could be connected to attention dysregulation. / 2021-10-07T00:00:00Z

No description available.

Through mutations in the genes TSC1 and TSC2, the genetic disorder Tuberous Sclerosis Complex (TSC) causes begin tumors to develop in different organs across the body. Of the many ways that this disorder can manifest, the brain is one of the most commonly affected organs in TSC. Mutations in TSC1 or TSC2 result in mTORC1 hyperactivation and can impact how the brain forms early in development. Most patients with TSC exhibit seizures and over half display some level of intellectual disability, highlighting the impact that mTORC1 hyperactivation can have on brain function and cognition. However, despite our understanding of the genetic cause of TSC, the mechanisms downstream of TSC1/TSC2 and mTORC1 that mediate TSC neuropathology are not well understood. Therefore, additional study of the cellular and molecular underlying the aberrant neurodevelopment found in TSC and other mTOR-overactivation disorders (collectively known as mTORopathies) is necessary for further understanding of these disorders. Of the pathways that have been identified to interact with mTORC1, there has been great interest in understanding the relationship between mTORC1 and Hippo-YAP/TAZ signaling. The Hippo pathway is an evolutionarily considered pathway that is crucial for regulating organ size through its control of the transcriptional co-activators YAP/TAZ. As exhibited through study of the murine brain, hyperactivation of YAP/TAZ causes changes in how the cortex develops, with several features overlapping with mTORC1 hyperactivation (including aberrant neuronal migration, changes in neuron structure, and increased progenitor proliferation). While the relationship between mTORC1 and YAP/TAZ has been explored in other systems, its connection in the brain has yet to be explored. In Chapter 1 of this dissertation, I first review how TSC affects cortical development as a whole by addressing what is known about the specific cell types and signaling pathways that are affected this disorder. Of the signaling pathways described, the Hippo- YAP/TAZ pathway is discussed in particular detail, addressing its role not only in the context of TSC and in terms of its interaction with mTORC1 signaling, but also in terms of its general role in cortical development. In discussing these studies, I describe the phenotypes seen in different mouse models and in the human brain, allowing for the identification of pathological features that are common between species and between different Cre lines. Following this initial review, I present our experimental aims, hypotheses, and experimental overview for this project in Chapter 2. In Chapter 3, I describe our investigation into the role of YAP/TAZ in the abnormal neurodevelopment that occurs in TSC. Through our analysis of human cortical tuber samples, I demonstrate that YAP/TAZ are elevated at the protein level and that two of their established target genes, CYR61 and CCN2, are elevated at the mRNA and protein levels. Having demonstrated that YAP/TAZ levels and activity are elevated in cortical tuber samples, I next went on to establish whether YAP/TAZ are similarly changed in our TSC animal model. Examination of Emx1-Cre driven Tsc2 cKO mice showed that the level of Yap/Taz were significantly elevated at E16.5. Having established that both YAP/TAZ levels are elevated in our animal model, I next sought to determine whether concurrent genetic manipulation of Yap/Taz in our Tsc2 cKO animals would reduce the severity of neuropathology seen in these mice. Triple conditional knockout (tcKO) of Yap/Taz/Tsc2 was sufficient to mitigate several features seen with mTORC1 hyperactivation in the brain, including the cortical thickness increases, abnormal neuronal migration in the cortex, hippocampal lamination defects, and hypomyelination found in their single Tsc2 cKO counterparts. Overall, these findings provide additional evidence that mTORC1 hyperactivation positively regulates YAP/TAZ. For the first time, this study describes elevation of YAP/TAZ in the brains of individuals with TSC and in the brains of a TSC mouse model. Furthermore, I provide evidence that reduction of Yap/Taz may have a beneficial effect on neuropathology in TSC, highlighting an area for future research in the development of novel therapeutics for this disorder. / Biomedical Sciences

No description available.

Epitranscriptomics, the study of RNA modifications, has become a hotspot of research over the last decade. Over 170 unique modifications have been discovered with a widespread occurrence in a diverse range of RNAs. 5-methylcytosine, m5C, is an evolutionarily conserved and reversable modification that regulates the stability and export of tRNAs, rRNAs, and mRNAs. m5C has recently been implicated in many biological phenomena including tumorigenesis, embryonic cell expansion and differentiation, brain development, and neuronal functions. While we are just beginning to understand the functions of m5C, a gold standard of m5C detection has yet to be established due to the low signal-to-noise presence of m5C. In this work, we utilize RNA bisulfite sequencing as a transcriptome-wide approach to understand the computational and chemical parameters needed to optimize m5C discovery in the mitochondria and the developing brain. In Chapter 1, we systematically evaluate four preparation conditions of bisulfite sequencing to identify potential presence of m5C-mRNAs localized to the mitochondria in neuronal stem cells. In tandem, we utilize unique molecular identifiers and a consortium of control template transcripts to evaluate sources of false positive m5C sites that may emerge from sequencing errors, PCR amplification, and the inadequate bisulfite conversion of transcripts. While improvements to mitochondrial transcript bisulfite conversion and false positive filtering were observed, no mitochondrial mRNAs were identified to be methylated, indicating no or very few methylated cytosines in mitochondrial mRNAs and the need for improved chemical methods to detect mitochondrial m5C-mRNAs if any. In Chapter 2, we employ the computational approaches established in Chapter 1 to survey the m5C landscape of the developing mammalian brain. We discover a general increase in unique m5C sites in mouse whole brain tissue when compared to neuronal cell cultures. Of these sites, we found the post-natal day 0 and 17 brain time points to undergo significant methylation level changes in comparison to the 6-week-old brain. These differentially methylated sites were significantly enriched for brain development, synaptic development, and transcriptional control gene network pathways. In Chapter 3, we expand on our findings in Chapter 2 to understand the impact of m5C reader FMRP and m5C eraser TET1 loss in the mouse post-natal day 17 brain. Among a set of m5C sites identified in wildtype or knockout samples, few were differentially methylated after protein ablation, suggesting m5C may rely on compensatory enzymes. Using FMRP-RNA pulldown assays to validate FMRP binding positions, we identified Ralbp1 to be hypermethylated and overexpressed in Fmr1-KO brain tissues. RalBP1 is a binding protein responsible for the endocytosis of AMPA receptors, a process critical for neuronal long term depression and brain development. / Doctor of Philosophy / Ribonucleic acid (RNA) is the product of deoxyribonucleic acid (DNA) transcription and the precursor to protein translation. Chemical modifications can be made to the bases of DNA, known as epigenetic modifications, to elicit new functions and responses to the environment. Epitranscriptomics refers to the study of RNA modifications that also serve unique roles and functions depending on the type of modification made. Here, we study the presence of 5-methylcytosine, a methyl group added to the cytosine (C) base of RNA. This modification is found throughout all branches of life and is known to promote the stability and export of many RNA types. Recently, studies have utilized many techniques including RNA bisulfite sequencing to find links between the presence of m5C-RNAs and cancer progression, stem cell development, and brain development. RNA bisulfite sequencing uses chemical applications to convert non-methylated "C"s to the RNA base "U", while retaining a "C" signature on methylated "C"s. However, due to the extremely low presence of RNA-m5C in comparison to DNA-m5C, sources of noise make it difficult to identify a true m5C signal. Because of this discrepancy, established analytical methods based on DNA biology may not be suitable for RNA analysis. To address shortcomings in current detection methods of RNA-m5C, we performed systematic analysis of 1) different preparation methods for improved m5C detection methods and 2) computational approaches for the filtering of false positive m5C sites, as described in Chapter 1. To achieve these goals, we expanded the breadth of analytical methods by including unique molecular identifiers and expanding the set of control RNA sequences to better grasp how false positive sites might be introduced into non-methylated sequences. While noticeable improvements were made to control RNA sequence false positive detection, we found that most mitochondrial RNAs did not carry the same m5C signatures as RNAs from other sources. Because of this difference, we could not conclude that mitochondrial mRNAs were methylated. Therefore, we suggest that future studies may need to develop better or alternative methods for the detection of mitochondrial RNA-m5Cs. In Chapters 2 and 3, we utilize the computational methods developed in Chapter 1 to understand how m5C levels change throughout the development of a mouse's brain. By investigating the m5C profiles of mouse newborn, young child, and juvenile brains, we found significant changes in m5C levels specific to certain RNAs. These RNAs are associated with neuronal growth, development, and maturation, which may have implications for m5C's role in cognitive development, intellectual disabilities, and neurodegenerative disorders. To discover if these RNAs could be affected by the absence of m5C-specific proteins, we created mice deficient in a protein m5C reader, FMRP, and an m5C eraser protein, TET1. Interestingly, we did not find a significant difference in mice deficient in the proteins, indicating m5C may rely on multiple proteins to serve redundant functions. However, one RNA, Ralbp1, was found to be significantly methylated in FMRP deficient models. This RNA is essential for developmental changes in the brain as well as neuronal growth and could be an interesting target for future research.

This thesis provides evidence for the presence of multiple RNAs in the axons and growth cones of developing thalamic cells, particularly the mRNA for the cell adhesion and Wnt-signalling-related molecule b-catenin. After many decades of effort, mRNAs have been shown to be present in the axons of many different systems in recent years. Furthermore, these mRNAs have been shown to be locally translated at the growth cone, and this local translation is required for axons to turn in response to multiple guidance cues. As studies accumulate, it is becoming clear that different axonal systems contain different complements of mRNAs and have different requirements for local translation. One axonal system which has not been investigated to date is the thalamocortical tract. The nuclei of the thalamus are connected to the areas of the cortex via bundles of axons which travel from the thalamus to the cortex via the ventral telencephalon during embyronic development. These axons make a number of turns and are guided by many cues and other axonal tracts before innervating their cortical target. In this thesis, a quantitative real-time polymerase chain reaction (qRT-PCR) approach is developed to isolate multiple mRNAs from developing thalamic axons in vitro, including b-catenin mRNA, b-actin mRNA, 18S ribosomal RNA and ten other mRNAs. The method used should be suitable for use with other axonal systems and also for testing the effect of guidance cues on mRNA expression in axons. The qRT-PCR results for b-catenin, b-actin and 18S have been validated using in situ hybridisation. Analysis of in situ hybridisation results indicates that b-catenin and 18S, but not b-actin, are upregulated in the growth cone compared to the axon. As b-catenin has been shown to be involved in axon guidance via Slit and ephrin guidance cues in other axonal systems, and these guidance cues act upon thalamocortical axons, the identification of b-catenin mRNA in thalamic axons is an important step towards a full understanding of the thalamocortical system. The results presented here indicate that local protein synthesis is likely to occur in thalamic axons as it does in other axonal systems, and that local translation is likely to be important for thalamic axonal responses to guidance cues and other axonal tracts.

L’objectif de cette thèse est de développer des méthodes IRM permettant d’étudier l’impact d’une ischémie focale transitoire sur le cerveau de rat nouveau-né. Les techniques utilisées sont l’imagerie à contraste renforcé par le manganèse (MEMRI), l’imagerie du tenseur de diffusion (DTI) ainsi que de façon préliminaire l’imagerie Q-ball (QBI). Le MEMRI après injection intra cérébrale a été utilisé afin d’étudier de manière dynamique le tractus cortico-thalamique, en parallèle le DTI a servi de marqueur de la structuration cérébrale. Les résultats ont montré une atteinte du tractus cortico-thalamique ipsi-latéral, sept et quatorze jours après ischémie. De manière générale le DTI a montré une structuration ralentie à la suite de l’ischémie. A partir de ces résultats la faisabilité d’une méthode d’acquisition rapide et de traitement de données Q-ball a été établie puis testée sur un animal immature. Les méthodes mises en place se sont révélées efficaces dans le suivi de la maturation cérébrale dans des conditions normales ainsi que pathologiques, ouvrant des perspectives d’études liées au développement cérébral. / The thesis aim is to develop MRI methods to study the impact of focal transient ischemia in neonatal rat brain. The principal techniques used are MEMRI (Manganese Enhanced MRI), DTI (Diffusion Tensor Imaging) and QBI (Q-Ball Imaging). MEMRI was used to observe in a dynamic way the cortico-thalamic manganese transport combined with the structural informations extracted from the DTI experiments. Results have shown a cortico-thalamic pathway disturbance, at seven and fourteen days after ischemia. Globally DTI results have shown a slowed brain structuration. From these results, the feasibility of a fast acquisition method and the post processing steps of Q-ball protocol was established and applied in an immature rat. The different MRI protocols developed during this thesis have shown good efficiency to follow the rat brain maturation, in healthy and pathological conditions, thus opening new perspectives for brain development studies.

The relationship between personality trait change and major life events is currently undergoing extensive investigations within the field of personality psychology. A debate has risen regarding whether or not major life events can bring about trait change, and how typical trait change patterns over the adult lifespan can be explained. It is valuable to understand how traits change because they predict important future outcomes. The Five-Factor Theory described by McCrae and Costa (2008a) states that traits are purely biological entities, and trait change is explained to result from processes of intrinsic biological maturation, unaffected by life events. This thesis reviewed the literature regarding the relationship of trait change and life events, and the research of potential biological bases of traits in the brain together with a brain developmental perspective of intrinsic maturation. Gaining an insight in the relationship between personality traits and the brain is a goal within a young field of research called personality neuroscience, and an agenda of the Five-Factor Theory. Major life events do cause trait change, but the relationship is complex. A brain developmental perspective of intrinsic maturation did not entirely correspond with patterns of typical trait change in young adulthood. The Five-Factor Theory is challenged and modifications are suggested. Neurobiological correlates of five-factor traits reveal issues and potentials for future research.