This thesis deals with the highly potent olfactory mucosa toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO2) and its non-toxic 2,5-chlorinated isomer (2,5-diClPh-MeSO2). In mice, both substances bind firmly in the olfactory mucosa and the olfactory bulb, which are important components of the sensory system. The 2,6-isomer induces olfactory mucosal necrosis with permanent loss of olfactory neuroepithelium and olfactory nerves. A major objective was to clarify the cause of this isomer-specific toxicity, and to identify which physicochemical characteristics determine the olfactory toxicity. The neurobehavioural toxicity of these substances was also examined. The results revealed a rapid CYP-catalysed covalent binding of 2,6-diClPh-MeSO2 in the rat olfactory mucosa, whereas the 2,5-dichlorinated isomer was not covalently bound. Acute and chronic olfactory mucosal pathology were investigated and compared in rats and mice. Twenty-four hours after dosing to rats, 2,6-diClPh-MeSO2 induced Bowman’s glands necrosis and sloughing of the olfactory epithelium similar to that previously reported in mice. At 3 weeks, however, there were dramatic differences in histological lesions. In mice, large parts of olfactory epithelium were replaced by respiratory-like epithelium. Large, bilateral, fibrous, cartilage and bone containing polyps occluding the lumen were confirmed. In rats, only minor patches of olfactory epithelium were replaced by a metaplastic atypical respiratory-like epithelium. 2,5-diClPh-MeSO2 was non-toxic in rats as well as in mice. In mice, 2,6-diClPh-MeSO2 induced a dose-dependent and long-lasting ( ≥12 weeks) hyperactivity as well as long-lasting maze learning deficits. At 2 weeks hyperactivity and maze learning deficits were observed also in rats. Unexpectedly, 2,5-diClPh-MeSO2 induced hyperactivity that lasted for two weeks. No effect on maze learning was observed with this isomer. No major differences between male and female rats or mice were found. In conclusion, the results show that a CYP-catalysed formation and covalent binding of a reactive 2,6-diClPh-MeSO2-metabolite in the Bowman’s glands precede the high olfactory mucosal toxicity in rodents. As determined by QSAR-modelling, a 2,6-dichlorinated benzene derivative with a large, polar, and strong electron withdrawing substituent in the primary position has the potential of being an olfactory mucosal toxicant. The observed 2,6-diClPh-MeSO2-induced increase in motor activity, and maze learning deficits, were not correlated to the olfactory mucosal lesions. I propose that 2,6-diClPh-MeSO2 causes a direct effect in the brain leading to neurobehaviuoral deficits.

<p>Altered dietary availability of the vital nutrient choline during early development leads to persistent changes in brain and behavior throughout adulthood. Prenatal choline supplementation during embryonic days (ED) 12-17 of the rodent gestation period enhances memory capacity and precision and hippocampal plasticity in adulthood, and protects against spatial learning and memory deficits shortly after excitotoxic seizures, whereas prenatal choline deficiency can compromise hippocampal memory and plasticity in adulthood. Recent evidence from our laboratory has determined that lifelong proliferation of newborn neurons in the adult hippocampus, a feature of adult hippocampal plasticity that has been implicated in some aspects of learning and memory, is modulated by early choline availability. Prenatal choline's effects on adult neurogenesis may be one mechanism for diet-induced cognitive changes throughout life and in response to injury, although little is known about the mechanisms underlying how prenatal choline alters adult neurogenesis or the neural mechanisms underlying prenatal choline supplementation's protection against cognitive deficits after seizures. To address these issues, the present set of experiments investigated how prenatal choline availability modulates specific properties of neurogenesis in the adult brain (in the intact brain and in response to injury), as well as hippocampal markers known to change in response to excitotoxin-induced seizures, and sought to relate changes in neurogenesis and in neuropathological markers following injury to changes in performance on spatial learning and memory tasks. Subjects in each experiment were adult offspring from rat dams that received either a control diet or diet supplemented with choline chloride or deficient of choline on ED 12-17. To measure neurogenesis, rats were given injections of the mitotic marker bromodeoxyurdine to label dividing cells in the hippocampus. Prenatal choline supplementation enhanced several properties of basal adult hippocampal neurogenesis (cell division and survival, neural stem/progenitor cell phenotype and proliferative capacity, trophic support), and this increase was associated with improvements in spatial working memory retention in a delayed-matching-to-place water maze task. In contrast, prenatal choline deficiency had little effect on basal adult hippocampal neurogenesis, and no effect on spatial memory performance. Prenatal choline supplementation also enhanced olfactory bulb neurogenesis without altering cell proliferation in the subventricular zone, while prenatal choline deficiency had no effect on either measure, showing for the first time that prenatal choline's effects on adult neurogenesis is similarly expressed in another distinct neurogenic region of the adult brain. Altered prenatal choline availability also modulated the hippocampal response to kainic acid-induced seizures where supplementation attenuated while deficiency had no effect on the injury-induced proliferative response of the dentate gyrus shortly after injury. Prenatal choline supplementation also attenuated other markers of hippocampal neuropathology shortly after seizures and promoted the long-term hippocampal recovery from seizures months after injury, including rescuing declines in adult hippocampal neurogenesis and in spatial memory performance in a standard water maze task. Taken together, these findings demonstrate a robust neuroprotective effect of prenatal choline supplementation that may be driven by enhanced adult hippocampal plasticity and trophic support prior to injury, and shed light on the mechanisms underlying how prenatal choline availability alters adult hippocampal neurogenesis, which may contribute to changes in memory capacity and precision both throughout life and following neural assault.</p> / Dissertation

<p>The olfactory system is believed to be the oldest sensory system. It developed to detect and analyse chemical information in the form of odours, and its organisation follows the same principles in almost all living animals - insects as well as mammals. Likely, the similarities are due to parallel evolution - the same type of organisation has arisen more than once. Therefore, the olfactory system is often assumed to be close to optimally designed for its tasks. Paradoxically, the workings of the olfactory system are not yet well known, although several milestone discoveries have been made during the last decades. The most well-known is probably the disovery of the olfactory receptor gene family, announced in 1991 by Linda Buck and Richard Axel. For this and subsequent work, they were awarded a Nobel Prize Award in 2004. This achievement has been of immense value for both experimentalists and theorists, and forms the basis of the current understanding of olfaction. The olfactory system has long been a focus for scientific interest, both experimental and theoretical. Ever since the field of computational neuroscience was founded, the functions of the olfactory system have been investigated through computational modelling. In this thesis, I present the basis of a biologically realistic model of the olfactory system. Our goal is to be able to represent the whole olfactory system. We are not there yet, but we have some of the necessary building blocks; a model of the input from the olfactory receptor neuron population and a model of the olfactory bulb. Taking into account the reported variability of geometrical, electrical and receptor-dependent neuronal characteristics, we have been able to model the frequency response of a population of olfactory receptor neurons. By constructing several olfactory bulb models of different size, we have shown that the size of the bulb network has an impact on its ability to process noisy information. We have also, through biochemical modelling, investigated the behaviour of the enzyme CaMKII which is known to be critical for early olfactory adaptation (suppression of constant odour stimuli).</p> / <p>Luktsystemet anses allmänt vara det äldsta sensoriska systemet. Det utvecklades för att upptäcka och analysera kemisk information i form av lukter, och det är organiserat efter samma principer hos nästan alla djurarter: insekter så väl som däggdjur. Troligen beror likheterna på parallell evolution -- samma organisation har uppstått mer än en gång. Därför antas det ofta att luktsystemet är nära optimalt anpassat för sina arbetsuppgifter. Paradoxalt nog är luktsystemets arbetssätt ännu inte väl känt, även om flera banbrytande framsteg gjorts de senaste decennierna. Det mest välkända är nog upptäckten av genfamiljen av luktreceptorer, som tillkännagavs 1991 av Linda Buck och Rikard Axel. För detta och efterföljande arbete belönades de med Nobelpriset år 2004. Upptäckten har varit mycket värdefull för både experimentalister och teoretiker, och formar grunden för vår nuvarande förståelse av luktsystemet. Luktsystemet har länge varit ett fokus för vetenskapligt intresse, både experimentellt och teoretiskt. Ända sedan fältet beräkningsbiologi grundades har luktsystemet undersökts genom datormodellering. I denna avhandling presenterar jag grunden för en biologiskt realistisk modell av luktsystemet. Vårt mål är att kunna representera hela luktsystemet. Så långt har vi ännu inte nått, men vi har några av de nödvändiga byggstenarna: en modell av signalerna från populationen av luktreceptorceller, och en modell av luktbulben. Genom att ta hänsyn till nervcellernas rapporterade variationer i geometriska, elektriska och receptor-beroende karaktärsdrag har vi lyckats modellera svarsfrekvenserna från en population av luktreceptorceller. Genom att konstruera flera olika stora modeller av luktbulben har vi visat att storleken på luktbulbens cellnätverk påverkar dess förmåga att behandla brusig information. Vi har också, genom biokemisk modellering, undersökt beteendet hos enzymet CaMKII, som är kritiskt viktigt för adaptering (undertryckning av ständigt närvarande luktstimuli) i luktsystemet.</p>

The distribution of cell types and synapses is well characterized in the rodent olfactory bulb (OB), and from that plausible models of odor processing have been constructed. Individual olfactory sensory neurons (OSNs) express only 1 of ~1000 odorant receptors (ORs) and send their axons to specific synaptic targets in the OB glomerular neuropil. Each glomerulus is innervated exclusively by OSN axons expressing the same OR. The distribution of these glomeruli is conserved across animals, as is the numerical relationship between number of expressed ORs and number of glomeruli in the OB. Our objective is to extend such results to the level of the human OB to determine how its cellular and synaptic organization, and more specifically how the number and distribution of its glomeruli, compare to what has been elucidated in mice. As there are ~2,000 glomeruli for ~1,000 ORs in mice, we predicted ~700 glomeruli in humans based on the ~350 intact OR genes identified in the human through genomic studies. Using immunohistochemistry, the organization of cells and synapses in human OBs was evaluated and quantified. While the laminar structure of the OB is broadly conserved between species, in the human OB the laminar organization as well as additional structural features suggest a less rigorously organized OB than in rodents, perhaps suggesting that odor processing in the human OB may be less efficient than in mice. Of particular note, the total number of glomeruli in the human OB differs significantly from predicted and demonstrates a high degree of variability amongst specimens, thus far ranging from approximately 3000 - 9000/OB. These results indicate that the principles of OR-homotypic axon convergence developed from mouse studies may not be readily applicable to the human, and that central processing of odor signals in the human may differ from those characterized in the mouse.

For decades electrophysiologists have recorded and characterized the biophysical properties of a rich diversity of neuron types. This diversity of neuron types is critical for generating functionally important patterns of brain activity and implementing neural computations. In this thesis, I developed computational methods towards quantifying neuron diversity and applied these methods for understanding the functional implications of within-type neuron variability and across-type neuron diversity. First, I developed a means for defining the functional role of differences among neurons of the same type. Namely, I adapted statistical neuron models, termed generalized linear models, to precisely capture how the membranes of individual olfactory bulb mitral cells transform afferent stimuli to spiking responses. I then used computational simulations to construct virtual populations of biophysically variable mitral cells to study the functional implications of within-type neuron variability. I demonstrate that an intermediate amount of intrinsic variability enhances coding of noisy afferent stimuli by groups of biophysically variable mitral cells. These results suggest that within-type neuron variability, long considered to be a disadvantageous consequence of biological imprecision, may serve a functional role in the brain. Second, I developed a methodology for quantifying the rich electrophysiological diversity across the majority of the neuron types throughout the mammalian brain. Using semi-automated text-mining, I built a database, Neuro- Electro, of neuron type specific biophysical properties extracted from the primary research literature. This data is available at http://neuroelectro.org, which provides a publicly accessible interface where this information can be viewed. Though the extracted physiological data is highly variable across studies, I demonstrate that knowledge of article-specific experimental conditions can significantly explain the observed variance. By applying simple analyses to the dataset, I find that there exist 5-7 major neuron super-classes which segregate on the basis of known functional roles. Moreover, by integrating the NeuroElectro dataset with brain-wide gene expression data from the Allen Brain Atlas, I show that biophysically-based neuron classes correlate highly with patterns of gene expression among voltage gated ion channels and neurotransmitters. Furthermore, this work lays the conceptual and methodological foundations for substantially enhanced data sharing in neurophysiological investigations in the future.

Precursores neurais originados na zona subventricular (ZSV) de algumas espécies animais possuem uma rota de migração neuronal destinada ao bulbo olfatório principal (BOP), onde os neuroblastos migrantes se diferenciam em interneurônios. Esta corrente migratória é mantida na idade adulta. A compreensão de como se organiza na idade fetal é essencial para a compreensão geral e estabelecimento de novas terapias celulares. O objetivo deste estudo é caracterizar a composição celular e organização morfológica da ZSV e da corrente rostral migratória (CRM) em encéfalos de fetos caninos. A ZSV, CRM e BOP foram obtidos de fetos caninos de aproximadamente 57 dias de idade gestacional. O tecido foi analisado através de coloração de Nissl, método de imunohistoquímica de dupla marcação com duplacortina (DCX), fator de transcrição SOX2, proteína glial fibrilar ácida (GFAP), calbindina (CALB), calretinina (CALR) e tirosina-hidroxilase (TH). Foram feitas a análise relativa da expressão da imunorreatividade e análise quantitativa de colocalização celular, além do método de microscopia eletrônica de transmissão. Os resultados mostram que a ZSV dorsal possui células imunorreativas (ir) para o DCX ao longo da parede ventricular, dispostas tangencialmente e fileiras de células SOX2-ir foram encontradas na mesma orientação. A imunorreatividade de GFAP foi mais forte na ZSV dorsal e as células possuem fibras dirigidas tangencialmente adjacentes ao ventrículo lateral e fibras orientadas radialmente em direção ao córtex. A CRM de feto de cão tem início na ZSV anterior e segue caudalmente ao redor da cabeça do núcleo caudado e desce na vertical até se curvar rostralmente em direção ao BOP onde termina na camada de células granulares (CCG). A CRM tem aparência homogênea e densa e possui células positivas para o DCX nas porções iniciais e para SOX2 e GFAP por toda a extensão. Não houve células positivas para CALB, CALR e TH em nenhuma região da ZSV e CRM. No BOP, os resultados mostraram que a camada glomerular (CG) possui células imunorreativas a CALR, TH, SOX2 e GFAP. Na camada plexiforme externa (CPE) houve células imunorreativas a CALB, CALR, SOX2 e GFAP e na CCG, houve células imunorreativas a CALR, SOX2 e GFAP. Na análise de colocalização, foram encontrados na CG neurônios CALR que colocalizam com células SOX2 e uma baixa colocalização de neurônios TH e células SOX2. Na CPE, foi observado um baixo número de colocalização de neurônios CALR e CALB e na CCG, as células SOX2 colocalizam com os neurônios CALR. As conclusões mostram que o feto de cão possui uma CRM em direção BOP, com imunorreatividade celular para DCX, SOX2 e GFAP na ZSV e CRM e para CALB, CALR, TH, SOX2 e GFAP nas principais camadas do BOP / Neural precursors originated in the subventricular zone (SVZ) of some animal species have a migration route destined for main olfactory bulb (MOB), where migrants neuroblasts differentiate into olfactory interneurons. This migratory stream is maintained in adulthood. Understanding how it is organized in fetal age is essential for general understanding and establishment of new cell therapies. The aim of this study is characterize the cellular composition and morphological organization of the SVZ and rostral migratory stream (RMS) of brains of canine fetuses. The SVZ, RMS and MOB was obtained from canine fetuses of the approximately 57 gestacional days-old. The tissue was analyzed by Nissl staining and by immunohistochemical methods for double labelling with doublecortin (DCX), transcription factor SOX2, glial fibrillary acid protein (GFAP), calbindin (CALB), calretinin (CALR) and tyrosinehydroxylase (TH). Semiquantitative analysis of immunoreactivity and quantitative analysis of colocalization were realized, besides ultrastructural analysis by electron microscopy. The results show that in dorsal SVZ, DCX immunoreactive cells were found along the ventricular wall, arranged tangentially and lines of SOX2 cells were also found in the same orientation. The GFAP immunostaining is stronger in dorsal SVZ with tangentially directed fibers near the lateral ventricle and radially oriented fibers toward the cortex. The RMS of dog fetus begins at anterior SVZ and follows caudally around the head of the caudate nucleus and vertically descends to bend rostrally into the MOB, where it ends in the granular cell layer (GCL).The RMS have SOX2 positive cells on entire length, showing a homogeneous appearance and high cell density. There is no positive CALB cells or CALR in any region of the SVZ and RMS. The results of the MOB show that the glomerular layer (GL) there were cells immunoreactive to CALR, TH, SOX2 and GFAP. In the external plexiforme layer (EPL) there were immunoreactive cells for CALR, CALB, SOX2 and GFAP and, the GCL, the prevalence is higher for CALR neurons, SOX2-ir and GFAP-ir cells. In colocalization analysis, they were found a some CALR positive neurons in GL that colabeled with SOX2 cells and a low colocalization of TH neurons and SOX2 cells. In EPL, was observed a low colocalization number of CALR and CALB neurons and in GCL, SOX2 cells colabeled with CALR neurons. The conclusions show that the dog fetus has a RMS directed to the MOB, with cellular immunoreactivity for DCX, SOX2 and GFAP in the ZSV and RMS and cellular immunoreactivity for SOX2 CALB, CALR, TH and GFAP in main olfactory bulb layers

Les cellules periglomerulaires du bulbe olfactif conforment une population hétérogène avec des propriétés moléculaires, synaptiques, morphologiques et biophysiques diverses toujours étudiés de façon indépendante. Toutefois, cette diversité suggère que des groupes différents des cellules periglomerulaires pourraient avoir des rôles différents. Dans la première partie de ma thèse je cherche à associer, pour la première fois, différents marqueurs de la diversité des neurones periglomerulaires de façon à aider à comprendre les potentielles implications fonctionnelles que les cellules periglomerulaires pourraient avoir dans le traitement de l’information olfactive. Les cellules periglomerulaires reçoivent des courants inhibiteurs postsynaptiques mais les circuits responsables de cette inhibition restent méconnus. À l’aide des enregistrements électrophysiologiques dans des tranches aiguës horizontales de bulbe olfactif de souris et des techniques d’optogénétique je montre que des projections centrifuges GABAergiques en provenance du télencéphale basal modulent fortement l’inhibition des cellules periglomerulaires de type 2 ainsi que des cellules granulaires et des cellules à axone courtes. / In the olfactory bulb periglomerular cells form a heterogeneous population with diverse molecular, synaptic, morphological and biophysical properties that have always been considered independently and never explored together. However, such diversity suggests different functional implications. On the first part of this thesis, I aim to associate, for the first time, different markers of periglomerular diversity together to put in perspective the functional implications that differebt subgroups of these cells could exert in odor processing. Periglomerular cells receive inhibitory postsynaptic currents but the circuits mediating this inhibition remain poorly understood. Using a combination of patch-clamp recordings in mouse horizontal olfactory bulb slices and optogenetics I demonstrate that centrifugal GABAergic projections from the basal forebrain strongly mediate inhibition of type 2 periglomerular cells but also granule cells and deep short axon cells.

Les projections corticales de retour conduisent l'information vers des relais de traitement de l'information plus précoces. Elles sont essentielles pour la perception sensorielle. En ce qui concerne l'olfaction, l'information sensorielle est constituée d'une multitude de molécules odorantes, et c'est ce mélange complexe qui pénètre dans la cavité nasale. En fonction du contexte, c'est une partie ou une autre de cet ensemble de molécules qui va être importante d'un point de vue comportemental. Les signaux corticaux de retour permettraient de focaliser son attention sur les odeurs pertinentes de l'environnement. Au cours de mon doctorat, j'ai étudié le rôle de la signalisation inhibitrice GABAergique dans ces retours corticaux vers le bulbe olfactif, le premier relais de l'information olfactive. La première partie de mon travail a mis en évidence une modulation métabotropique GABAergique du retour cortical excitateur. Nos expériences caractérisent ensuite l'effet produit par cette modulation sur le bulbe olfactif. Nous avons ainsi démontré que la signalisation GABAergique au niveau de retours corticaux change de manière profonde la réponse du bulbe olfactif aux stimuli olfactifs. Dans un deuxième temps, j'ai trouvé que le cortex olfactif envoie non seulement des projections de retour excitatrices, mais aussi des retours inhibiteurs. Des expériences précisent ensuite la localisation de ce retour GABAergique, ainsi que son impact sur le bulbe olfactif. Nous avons notamment observé qu'en manipulant l'activité de ces fibres GABAergiques, nous pouvions modifier le comportement olfactif. / Cortical feedback conducts information towards earlier relays of information processing. It is instrumental for sensory perception. In the olfactory system, odorants are never experienced in isolation by the nose, and they might be meaningful to the animal or not depending on the context. Feedback inputs onto early processing stages are poised to permit selective attention to the relevant odorants in the olfactory scene. During my thesis work, I focused on understanding the key role that inhibitory GABAergic signaling plays in the cortical feedback to the olfactory bulb in mice.The first part of my work started with the discovery of excitatory transmission between cortical feedback inputs and the olfactory bulb is modulated by metabotropic receptors for GABA. Next, the impact of this regulation on the olfactory bulb network was investigated. We found that GABAergic signaling at cortical feedback axons profoundly changes the response of the olfactory bulb output cells to odor stimulation. In the second part of my thesis, I found that the cortical projections to the olfactory bulb not only comprises of excitatory components, but also inhibitory components. The precise origin of this GABAergic feedback was then determined and its impact on the olfactory bulb network is currently assessed. In particular, we observed that manipulating the activity of this GABAergic feedback perturbs olfactory behavior.

abstract: The increasing availability of experimental data and computational power have resulted in increasingly detailed and sophisticated models of brain structures. Biophysically realistic models allow detailed investigations of the mechanisms that operate within those structures. In this work, published mouse experimental data were synthesized to develop an extensible, open-source platform for modeling the mouse main olfactory bulb and other brain regions. A “virtual slice” model of a main olfactory bulb glomerular column that includes detailed models of tufted, mitral, and granule cells was created to investigate the underlying mechanisms of a gamma frequency oscillation pattern (“gamma fingerprint”) often observed in rodent bulbar local field potential recordings. The gamma fingerprint was reproduced by the model and a mechanistic hypothesis to explain aspects of the fingerprint was developed. A series of computational experiments tested the hypothesis. The results demonstrate the importance of interactions between electrical synapses, principal cell synaptic input strength differences, and granule cell inhibition in the formation of the gamma fingerprint. The model, data, results, and reproduction materials are accessible at https://github.com/justasb/olfactorybulb. The discussion includes a detailed description of mechanisms underlying the gamma fingerprint and how the model predictions can be tested experimentally. In summary, the modeling platform can be extended to include other types of cells, mechanisms and brain regions and can be used to investigate a wide range of experimentally testable hypotheses. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2019

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