Thesis (Ph. D.)--Virginia Commonwealth University, 2005. / Prepared for: Dept. of Pharmacology and Toxicology. Bibliography: leaves 142-153.

Schizophrenia is a neurodevelopmental disorder affecting 1% of the general population. Dysfunction of the prefrontal cortex (PFC) is associated with the etiology of schizophrenia. Moreover, a substantial deficit of GAD1mRNA in schizophrenic PFC has been reported by different groups. However, the underlying molecular mechanisms are still unclear. Interestingly, epigenetic factors such as histone modifications and DNA methylation could be involved in the pathogenesis of schizophrenia during the maturation of the PFC. In my work, I identified potential epigenetic changes in schizophrenic PFC and developmental changes of epigenetic marks in normal human PFC. Furthermore, mouse and neuronal precursor cell models were used to confirm and investigate the molecular mechanisms of the epigenetic changes in human PFC. My initial work examined whether chromatin immunoprecipitation can be applied to human postmortem brain. I used micrococcal nuclease (MNase)-digested chromatin instead of cross-linked and sonicated chromatin for further immunoprecipitation with specific anti-methyl histone antibodies. Surprisingly, the integrity of mono-nucleosomes was still maintained at least 30 hrs after death. Moreover, differences of histone methylation at different genomic loci were detectable and were preserved within a wide range of autolysis times and tissue pH values. Interestingly, MNase-treated chromatin is more efficient for subsequent immunoprecipitation than crosslinked and sonicated chromatin. During the second part of my dissertation work, I profiled histone methylation at GABAergic gene loci during human prefrontal development. Moreover, a microarray analysis was used to screen which histone methyltransferase (HMT) could be involved in histone methylation during human prefrontal development. Mixed-lineage leukemia 1 (MLL1), an HMT for methylation at histone H3 lysine 4 (H3K4), appears to be the best candidate after interpreting microarray results. Indeed, decreased methylation of histone H3 lysine 4 at a subset of GABAergic gene loci occurred in Mll1 mutant mice. Interestingly, clozapine, but not haloperidol, increased levels of trimethyl H3K4 (H3K4me3) and Mll1 occupancy at the GAD1 promoter. I profiled histone methylation and gene expression for GAD1 in schizophrenics and their matched controls. Interestingly, there are deficits of GAD1 mRNA levels and GAD1 H3K4me3 in female schizophrenics. Furthermore, I was also interested in whether the changes of GAD1 chromatin structure could contribute to cortical pathology in schizophrenics with GAD1 SNPs. Remarkably, homozygous risk alleles for schizophrenia at the 5’ end of the GAD1 gene are associated with a deficit of GAD1 mRNA levels together with decreased GAD1 H3K4me3 and increased GAD1H3K27me3 in schizophrenics. Finally, I shifted focus on whether DNA methylation at the GAD1 promoter could contribute to a deficit of GAD1 mRNA in schizophrenia. However, no reproducible techniques are available for extracting genomic DNA specifically from GABAergic neurons in human brain. Therefore, I used an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of active and silenced gene expression. The methylation frequencies of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined from two chromatin fractions (H3K4me3 and H3K27me3) separately. Consistently, the proximal promoter region of GAD1 is more resistant to methylation in comparison to intron 2 of GAD1 in either open or repressive chromatin fractions. Interestingly, overall higher levels of DNA methylation were seen in repressive chromatin than in open chromatin. Surprisingly, schizophrenic subjects showed a significant decrease of DNA methylation at the GAD1proximal promoter from repressive chromatin. Taken together, my work has advanced our knowledge of epigenetic mechanisms in human prefrontal development and the potential link to the etiology of schizophrenia. It could eventually provide a new approach for the treatment of schizophrenia, especially in the regulation of methylation at histone H3 lysine 4.

Studies using a rat model of prenatal protein malnutrition (PPM) followed by nutritional rehabilitation show that PPM produces changes in the brain and behavior that endure throughout adulthood. Early studies investigated the vulnerability of the hippocampus, a structure involved in learning and memory, and reported permanent anatomical, physiological, and functional alterations. However, PPM also produces deficits in attentional processes, suggesting vulnerability across a broader cortical network including the parahippocampal region (PHR) and the prefrontal cortex (PFC). This thesis investigates the anatomical, functional, and molecular alterations in these regions resulting from PPM. This was accomplished through 4 studies: 1) A quantitative assessment of the number of neurons in the PHR and in the PFC using design-based stereology; 2) An evaluation of the impact of the PPM on metabolic activity in the PFC using the metabolic marker 2-[14C]deoxyglucose (2DG); 3) The identification of specific neuronal subtypes differentially activated during restraint stress in the PPM network using double-labelling immunohistochemistry; 4) The quantification of mRNA and protein expression of KCNJ3 (GIRK1), a potassium channel involved in regulating neural excitability, using quantitative polymerase chain reaction and Western blot analysis. Results showed that: 1) Neuron number in the PFC is unchanged by PPM, but two subfields of the PHR, the presubiculum and medial entorhinal cortex, exhibit significantly lower numbers in PPM rats; 2) Metabolic activity in specific PFC regions associated with attention including the prelimbic, infralimbic, anterior cingulate, and orbitofrontal cortices was reduced relative to controls while other regions, such as the hippocampus, were unaffected; 3) Exposure to stress evokes a significant increase in the number of inhibitory interneurons that are activated in the PFC of PPM rats which could likely contribute to the observed overall reduction in PFC activity; 4) For the KCNJ3 channel, PPM induces lower levels of mRNA and protein expression in the PFC while levels in the hippocampus and brain stem/basal ganglia are unchanged. Together, these data show that PPM creates permanent anatomical, functional, and molecular alterations selective to specific subfields, cell types, and molecules leading to an imbalance between excitatory and inhibitory processes in the PHR-PFC network of adult rats.

The motor symptoms of Parkinson's disease (PD) have been linked to the emergence of exaggerated oscillatory activity in the 13 - 35 Hz beta range in recordings of the basal ganglia (BG) thalamocortical circuit of PD patients and animal models. PD patients and animal models also express dopamine-dependent cognitive impairments, implying effects of dopamine loss on the function of the anterior cingulate cortex (ACC). This thesis examines the electrophysiological behavior of the BG thalamocortical circuit in PD and dopamine-normal states during cognitive and motor activity. In vivo recordings in the BG of PD and dystonic patients were used to study the influence of dopamine during a test of executive function. Normal executive function was also investigated in the dopamine-healthy ACC of chronic pain patients. Both the BG and ACC exhibited lateralized electrophysiological responses to feedback valence. The BG also exhibited dopamine-sensitive event-related behavior. In additional experiments, chronically implanted recording electrodes in awake, behaving hemiparkinsonian rats were used to examine the transmission of synchronized oscillatory activity from the BG, through the ventral medial (VM) thalamus, to the ACC. Modulation of subthalamic nucleus, VM thalamus, and ACC activity during a simple cognitive/movement task was also investigated in hemiparkinsonian rats. Findings in the rat model suggest that ACC-mediated executive function is dopamine-sensitive and is reflected in the region's electrophysiology. These results may provide further insight into the significance of excessive oscillatory activity in PD and its influence on cognitive systems.

Traumatic incidences happen all around the globe. Some of the people who experience trauma develop post-traumatic stress disorder (PTSD), while some do not. Even more interesting is that some also experience growth afterwards (post-traumatic growth; PTG). The purpose of this paper is to look at neural aspects of why some people develop PTSD and others PTG after a traumatic event. To fulfill the aim, both PTSD and PTG will be reviewed to create an image of the existing research in behavioral and neurological terms. In addition to looking at the constructs separately, a chapter will also look at studies where both PTSD and PTG are acknowledged collaterally in participants. When looking deeper into the theories of PTSD divisions occur, and more research is needed to establish the most prominent explanation of PTSD. PTG on the other hand has only been studied for a short period of time but yields important insights into trauma-related outcomes. These fields need to be submerged and new multidisciplinary definitions are needed for future research. The key to PTSD is suggested to emerge within the new field.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Kristýna Ludínova Supervisor: PharmDr. Jan Zitko, Ph.D. External supervisors: Dr. Manfred Oswald, Prof. Dr. Rohini Kuner Title of diploma thesis: Delineating Pain and Fear Engrams in the Prefrontal Cortex Pain is a complex process associated with activation of various brain centres. According to evidence of imaging studies in humans and rodents the medial prefrontal cortex (mPFC) ranks amongst the brain area consistently activated during painful perception. The mPFC circuits underlies functionally-distinct processes, such as pain, emotional response, decision-making, attention amongst others. However, the precise contribution of mPFC in pain related function remains to be unknown. The aim of the study was to delineate how pain and fear are manifested at the cellular level within the regions of PFC. By employing activity dependent neuronal labelling we tested if cellular ensembles activated in pain and fear behaviours within the mPFC are distinct. We investigated a potential use of activity-dependent DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) expression in order to test for the functional role of PFC ensembles in pain and fear behaviour. Our...

La stimulation magnétique transcranienne (SMT) est une technique non invasive qui permet de stimuler le cerveau. Les SMT répétitives (SMTr), c'est-à-dire l'application de nombreuses impulsions magnétiques, sont capable d'induire des modifications de longue durée de l'excitabilité neuronale. La SMT s'est développée dans un but thérapeutique et scientifique. Les effets après la SMTr sur le cortex moteur sont bien documentés chez les individus sains, mais on en sait moins sur la stimulation du cortex préfrontal dorso-latéral (DLPFC).L'objectif de cette thèse était de comparer différents protocoles SMTr sur des sujets sains et de trouver des marqueurs électroencéphalograpiques (EEG) de la réponse ou pas à la thérapie SMTr dans la dépression majeure et bipolaire. La principale originalité de la méthode présentée est la comparaison intra-sujet d'effets entre-protocoles et le développement de techniques de localisation de sources.Nous avons étudié chez 20 sujets sains comment les oscillations corticales sont modulées suite à quatre protocoles SMTr actifs différents, et à un protocole sham utilisé comme contrôle, du DLPFC gauche et en comparant la puissance spectrale d'EEG avant et après SMTr de durée de 15 minutes. Le spectre EEG a été estimé grâce à la transformée de Fourier rapide (FFT) et partitionné en bandes de fréquence selon la classification commune.Nous avons trouvé pour chaque protocole actif une diminution significative de puissance delta et theta sur les électrodes préfrontales gauches, principalement localisées dans le DLPFC gauche. Dans des bandes de fréquences plus hautes, la diminution de puissance dans le DLPFC a été de plus observée dans le DLFPC controlatéral et dépend du protocole de stimulation. Parce que les activités delta et theta sont généralement associées à l'inhibition corticale, ces résultats suggérent que la SMTr du DLPFC diminue transitoirement l'inhibition corticale locale. Aussi, les oscillations d'EEG rapides sont associées à l'excitabilité corticale et on peut conclure que des diminutions observées non spécifiques dans l'activité rapide localisée dans le DLPFC suggérent également une réduction de l'excitabilité corticale.Dans la deuxième expérience, nous avons travaillé sur groupe de patients, souffrant de trouble dépressif majeur (MDD) et de trouble bipolaire (BP). Dans cette étude ouverte, nous avons cherché à déterminer s'il existe des différences d'EEG de repos dans l'activité cérébrale entre patients BP et MDD, et entre les répondeurs et non-répondeurs à la SMTr à 10 Hz en étudiant des biomarqueurs d'EEG. Le protocole SMTr à 10 Hz étaient le même entre patients MDD et BP. Les propriétés EEG dans les deux troubles dépressifs ont été étudiées, en comparant la puissance spectrale des enregistrements pré- et post-SMTr EEG au cours des sessions thérapeutiques chez les patients répondeurs et non-répondeurs.La conclusion est qu'il est possible de distinguer les répondeurs des non-répondeurs au traitement SMTr. Les répondeurs avaient une puissance en basse fréquence plus importante. Une augmentation de puissance alpha a aussi été observée au niveau du cortex cingulaire ventral dans les deux groupes. La comparaison des MDD et BP a révélé une activité significativement plus élevée dans la puissance des bandes thêta et bêta chez les patients BP, principalement localisée dans le cortex préfrontal. / Transcranial magnetic stimulation (TMS) is a non-invasive technique for stimulating the brain. A brief electric current passing through a magnetic coil produces a brief, high-intensity magnetic field which stimulates the brain. Repetitive TMS, application of many magnetic pulses, is able to induce relatively long-lasting excitability changes and nowadays is being developed for various therapeutic and scientific purposes. The after-effects of rTMS over motor cortex are well documented in healthy individuals, however less is known about the stimulation of dorso-lateral prefrontal cortex (DLPFC). The aim of this PhD thesis was to compare different rTMS protocols in healthy subjects and to find neurophysiological EEG biomarkers characteristic for response or not to rTMS therapy in major and bipolar depression. The main originality of presented method is within-subject comparison of between-protocols effects. Additionally, source localisation was performed in both analyses. Here, we studied in 20 healthy subjects how cortical oscillations are modulated by four different active rTMS protocols (1 Hz, 10 Hz, cTBS and iTBS) of the left DLPFC, and by a sham-1Hz protocol used as a control condition, by comparing the spectral power of pre- and post-rTMS electroencephalographic (EEG) recordings of 15 minutes duration. EEG spectrum was estimated with the Fast Fourier transform (FFT) and partitioned using the common physiological frequency. We found for every active protocol a significant decrease of delta and theta power on left prefrontal electrodes, mainly localised in the left DLPFC. In higher frequency bands (beta and gamma), the decrease of power in the DLPFC was also observed additionally in the contralateral DLFPC and depended on the stimulation protocol. Because large delta and theta activity is usually associated with cortical inhibition, these results suggest that rTMS of DLPFC transiently decreases local cortical inhibition. Furthermore, fast EEG oscillations are associated to cortical excitability and it can be concluded that observed decreases in fast activity, unspecific to protocol, localised in the DLPFC also suggest reduced cortical excitability, which accompanies a decrease in cortical inhibition. In the second experiment we worked on two subgroups of patients, with major depressive disorder (MDD) and bipolar disorder (BP). In this open study we aimed to examine whether there are EEG differences in resting brain activity between BP and MADD patients, and between responders and non-responders to 10 Hz repetitive transcranial magnetic stimulation (rTMS) by studying EEG biomarkers. Eight MDD (6 females) and 10 BP patients (6 females) were included. The 10 Hz rTMS protocol was the same for MDD and BP. The different patterns of EEG activity in both depressive disorders were studied, by comparing spectral power of pre- and post-rTMS EEG recordings throughout the therapeutic sessions in responders and non-responders.The most important finding is that it is possible to distinguish responders from non-responders to the rTMS treatment. Responders showed significantly higher power of low frequencies. Therefore, an increase of alpha power was observed in ventral cingulate cortex in both groups. The comparison of MDD and BP disorders revealed significantly higher activity in theta and beta bands power in BP patients, mainly localised in prefrontal cortex.

Le cortex préfrontal (cpf) est à la base des processus cognitifs supérieurs qui sont modulés majoritairement par des entrées cholinergiques via les récepteurs nicotiniques de l'acétylcholine (nachrs). cette région du cerveau présente " par défaut " une activité spontanée, qui est modifiée dans le cas de troubles psychiatriques, tels que la schizophrénie, et de maladies neurodégénératives comme la maladie d'alzheimer. des études de génétique humaine ont mis en évidence la nature polymorphique de gènes spécifiques codant pour les nachr qui augmentent les risques de tabagisme et de schizophrénie. de nombreux laboratoires, dont le notre, ont montré que les souris ayant une altération de la fonction du gène nachr présentent des déficits comportementaux cpf-dépendants. cependant, la manière dont les polymorphismes humains correspondants altèrent les mécanismes cellulaires et circuits sous-jacents aux comportements reste inconnue. de ce fait, nous avons donc développé et étudié des modèles de souris liés à la schizophrénie, à la dépendance à la nicotine et à la maladie d'alzheimer. l'utilisation in vivo de l'imagerie bi-photon au niveau du cpf de souris éveillées et de souris anesthésiées a montré que différentes sous-unités nachr sont impliquées dans le contrôle de l'activité spontanée du cortex préfrontal, via un circuit d'inhibition hiérarchique. de plus, l'effet de l'administration chronique de nicotine sur l'activité cérébrale a été étudié, en fournissant des concentrations analogues à celles observées chez des fumeurs. ce travail met en lumière le rôle de la neurotransmission cholinergique dans l'orchestration des fonctions cognitives. / The prefrontal cortex (pfc) underlies higher cognitive processes that are modulated by cholinergic inputs largely via nicotinic acetylcholine receptors (nachrs). this brain region exhibits spontaneous “default” activity, which is altered in neuropsychiatric disorders, such as schizophrenia (scz), and neurodegenerative diseases such as alzheimer’s disease (ad). both of these disorders have a strong impact and burden on society. human genetic studies have highlighted the polymorphic nature of specific nachrs genes that increase risk for smoking and scz. several laboratories, including our own, have shown that mice with altered nachr gene function exhibit pfc-dependent behavioral deficits, but how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying the behaviors is unknown. here, mouse models related to scz, nicotine dependence and ad were developed and studied. using in vivo two-photon imaging in the pfc of both awake and anesthetized mice, different nachr subunits were shown to control spontaneous pfc activity through a hierarchical inhibitory circuit. furthermore, the effect of chronic nicotine administration on brain activity, by delivering concentrations analogous to that observed in smokers, was studied. the impact of nicotine on pfc layer ii/iii microcircuits altered in pathology can be extended to therapeutic strategies with strong candidates being positive allosteric modulators (pams) for defined nachr subunits. we hope that this work sheds light on the role of cholinergic neurotransmission in the orchestration of cognitive functions and will inspire further research in this direction.

abstract: The brain is a fundamental target of the stress response that promotes adaptation and survival but the repeated activation of the stress response has the potential alter cognition, emotion, and motivation, key functions of the limbic system. Three structures of the limbic system in particular, the hippocampus, medial prefrontal cortex (mPFC), and amygdala, are of special interest due to documented structural changes and their implication in post-traumatic stress disorder (PTSD). One of many notable chronic stress-induced changes include dendritic arbor restructuring, which reflect plasticity patterns in parallel with the direction of alterations observed in functional imaging studies in PTSD patients. For instance, chronic stress produces dendritic retraction in the hippocampus and mPFC, but dendritic hypertrophy in the amygdala, consistent with functional imaging in patients with PTSD. Some have hypothesized that these limbic region's modifications contribute to one's susceptibility to develop PTSD following a traumatic event. Consequently, we used a familiar chronic stress procedure in a rat model to create a vulnerable brain that might develop traits consistent with PTSD when presented with a challenge. In adult male rats, chronic stress by wire mesh restraint (6h/d/21d) was followed by a variety of behavioral tasks including radial arm water maze (RAWM), fear conditioning and extinction, and fear memory reconsolidation to determine chronic stress effects on behaviors mediated by these limbic structures. In chapter 2, we corroborated past findings that chronic stress caused hippocampal CA3 dendritic retraction. Importantly, we present new findings that CA3 dendritic retraction corresponded with poor spatial memory in the RAWM and that these outcomes reversed after a recovery period. In chapter 3, we also showed that chronic stress impaired mPFC-mediated extinction memory, findings that others have reported. Using carefully assessed behavior, we present new findings that chronic stress impacted nonassociative fear by enhancing contextual fear during extinction that generalized to a new context. Moreover, the generalization behavior corresponded with enhanced functional activation in the hippocampus and amygdala during fear extinction memory retrieval. In chapter 5, we showed for the first time that chronic stress enhanced amygdala functional activation during fear memory retrieval, i.e., reactivation. Moreover, these enhanced fear memories were resistant to protein synthesis interference to disrupt a previously formed memory, called reconsolidation in a novel attempt to weaken chronic stress enhanced traumatic memory. Collectively, these studies demonstrated the plastic and dynamic effects of chronic stress on limbic neurocircuitry implicated in PTSD. We showed that chronic stress created a structural and functional imbalance across the hippocampus, mPFC, and amygdala, which lead to a PTSD-like phenotype with persistent and exaggerated fear following fear conditioning. These behavioral disruptions in conjunction with morphological and functional imaging data reflect a chronic stress-induced imbalance between hippocampal and mPFC regulation in favor of amygdala function overdrive, and supports a novel approach for traumatic memory processing in PTSD. / Dissertation/Thesis / Ph.D. Psychology 2013

La catégorisation est un ensemble d’opérations mentales qui permettent de classer les objets et les évènements. C’est un processus crucial pour de nombreuses situations, telles que la survie dans le monde animal, l’apprentissage chez l’enfant, ou encore le raisonnement abstrait et la résolution de problèmes. Les patients ayant des lésions du cortex préfrontal présentent des difficultés pour les tâches de catégorisation, et l’existence de ces difficultés est corrélée au handicap fonctionnel de ces patients. Dans une première partie, nous avons mis au point une tâche de catégorisation adaptée pour l’utilisation chez le patient, intitulée SimiCat. A l’aide de cette tâche, nous avons précisé les difficultés de catégorisation des patients et montré que les erreurs de type différenciations sont spécifiques des patients frontaux. La tâche SimiCat présente une très bonne valeur diagnostique pour distinguer les patients ayant une démence fronto-temporale de ceux ayant une maladie d’Alzheimer. Dans une deuxième partie, nous avons utilisé l’IRM fonctionnelle pour préciser les bases cérébrales de deux processus clés pour la catégorisation : l’abstraction et la détection de similitudes. Nous avons montré que l’abstraction repose sur le cortex préfrontal dorsolatéral gauche, alors que la détection de similitudes repose sur le cortex préfrontal ventrolatéral bilatéral. A l’aide de la morphométrie basée sur le voxel, nous avons montré que la variabilité des performances de catégorisation des sujets sains était corrélée au volume de la portion antérieure du gyrus temporal moyen et inférieur droit, avec un gradient postéro-antérieur selon le niveau d’abstraction de la catégorisation. Dans une troisième partie, nous avons mis au point une tâche de double amorçage sémantique que nous utiliserons pour étudier les processus de catégorisation automatique chez les patients ayant des lésions frontales et temporales. A partir de ces résultats, nous proposons un modèle d’organisation cérébrale pour la catégorisation, reposant sur les régions temporales antérieures, le cortex préfrontal ventrolatéral bilatéral et dorsolatéral gauche. Nos résultats permettent également de mieux comprendre les déficits de catégorisation des patients, ce qui permettra d’adapter leur prise en charge diagnostique et thérapeutique. / Categorization is a set of mental processes that allow classifying objects and events. It is crucial in various contexts such as survival in animals, concept learning in children, abstract reasoning and problem solving. Patients with brain lesions involving the prefrontal cortex are impaired in categorization tasks. Categorization impairment correlates with functional autonomy in dementia. In the first part, we have developed a task, named SimiCat that we designed in order to assess categorization abilities in patients. With the help of this task, we showed that differentiation errors are specific of frontal patients. The SimiCat task has a good diagnostic value to distinguish behavior variant Frontotemporal dementia from Alzheimer disease.In the second part, we assessed the brain correlates of categorization. With functional MRI, we showed that abstraction involves the left dorsolateral prefrontal cortex, while similarity detection involves bilateral ventrolateral prefrontal cortex. With voxel-based morphometry we showed that variability in categorization performances correlates with the volume of the right anterior temporal lobe, with a caudo-rostral gradient according to abstraction. In the third part, we developed a double priming task that we will use to assess automatic categorization processes in patients with temporal and frontal lesions. Based on these results, we propose a model of brain organization for categorization. This model involves both anterior temporal lobes, as well as bilateral ventrolateral and left dorsolateral prefrontal cortices. Our results also contribute to a better understanding and management of patients suffering from categorization deficits.