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The mitral cells of the accessory olfactory bulb (AOB) of anuran frogs project their axons directly to the medial amygdala (MeA) along the accessory olfactory tract. An en bloc preparation of the telencephalon of the American bullfrog Lithobates catesbeiana was utilized to study a form of low-frequency inducible long-term potentiation (LTP) expressed at the synapse formed between the terminals of the accessory olfactory tract and the neurons of the MeA. Delivery of repetitive 1Hz-stimulation or sets of 5Hz tetani to the accessory olfactory tract both induced potentiation that was stable for over an hour, as measured by extracellular field recordings. LTP induced by 5Hz tetanus was associated with a decrease in paired-pulse ratio, which would be consistent with an increased probability of release contributing to the increased synaptic strength. Blockade of neither NMDA nor kainate glutamate receptors, with AP5 and UBP310 respectively, prevented LTP induction by 5Hz tetanus; however expression of LTP was partially masked in the presence of UBP310. These results suggest that kainate receptors are involved in the expression of LTP at the AOB-MeA synapse, though the means by which LTP is induced remains unclear. / Graduate / 2016-09-28

Olfactory receptor neurons (ORNs) maintain the ability to regenerate. These neurons reside in the olfactory epithelium and project axons that connect to the olfactory bulbs. Despite the diffuse distribution of ORNs in the olfactory epithelium, they converge at discrete glomeruli in the olfactory bulb. In the P2 IRES tau-lacZ mouse, the P2 ORN subtype has been previously mapped to two glomeruli, using X-gal staining. To determine if age affects ORN regeneration, left olfactory nerve transections were performed on P2 mice from immature (five-weeks old) and mature (1 6-weeks old) groups. Following recovery, the olfactory bulbs were processed to observe ORN regeneration. A significant difference was seen in the number and mapping of full P2 glomeruli between lesioned and control olfactory bulbs, but not between the age groups. This suggests that age differences between the two groups in this study were not large enough to affect the regeneration of P2 ORNs.

Os odores são detectados por uma grande família de receptores olfatórios (ORs) que são expressos nos neurônios olfatórios localizados no nariz. Os ORs ativados por um determinado odor acoplam-se à proteína Galfaolf que irá promover a ativação da adenilil ciclase III, resultando na produção de AMPc. O aumento da concentração de AMPc irá ativar canais iônicos dependentes de AMPc, tendo como consequência a despolarização do neurônio olfatório. A informação desencadeada pela ativação de determinados ORs é então transmitida para regiões específicas do cérebro promovendo a percepção do odor. A determinação da especificidade dos ORs para diferentes odores irá contribuir para o entendimento de como os odores são discriminados pelo sistema olfatório, entretanto, poucos ORs tiveram seus ligantes definidos devido a dificuldade de expressão funcional de ORs em sistema heterólogo. Em nosso trabalho, utilizamos o sistema de duplo-híbrido em levedura a fim de determinar potenciais novos reguladores para Galfaolf. Deste experimento, identificamos que Ric-8B (Ric, abreviatura de Resistant to Inhibitors of Cholinesterase), um provável GEF (GTP Exchange Factor), é capaz de interagir com Gaolf. Assim como Gaolf, Ric-8B é predominantemente expresso nos neurônios olfatórios maduros e em regiões específicas do cérebro. A restrita co-localização de Gaolf e Ric-8B fortemente indica que Ric-8B é uma proteína que participa da via de transdução de sinal de Galfaolf. Através de nossos ensaios, utilizando células HEK-293, foi possível mostrar que Ric-8B é capaz de potencializar a atividade de Galfaolf, tendo como consequência o aumento da produção de AMPc em sistema heterólogo. Por fim, nós mostramos que Ric-8B é capaz de promover a expressão funcional de ORs em sistema heterólogo. Nossos resultados demonstram que a expressão de Ric-8B é capaz de aumentar o acúmulo de Galfaolf na periferia de células HEK-293T, indicando que Ric-8B promove a expressão funcional de ORs provavelmente através da melhora da eficiência do acoplamento dos ORs com Galfaolf. Nossos resultados demonstram que o uso de Ric-8B em um sistema em larga escala irá permitir a expressão funcional de diversos ORs, permitindo a identificação de seus respectivos ligantes. Tal análise irá contribuir para o melhor entendimento do mecanismo de percepção dos odores. / Odorants are detected by a large family of odorant receptors (ORs) expressed in the olfactory neurons in the nose. The activated receptors couple to an olfactory-specific G-protein (Galphaolf), which activates adenylyl cyclase III to produce cAMP. Increased cAMP levels activate cyclic nucleotide-gated channels, causing cell membrane depolarization. The information provided by the odorant receptors is transmitted to specific regions of the brain leading to odorant perception. The determination of the odorant specificities of the different ORs will contribute to the understanding of how odorants are discriminated by the olfactory system. However, only a few ORs have been linked to odorants they recognized to date because ORs are not efficiently expressed in heterologous cells since they are poorly expressed on the cell surface. Here we used yeast two-hybrid to search for potential regulators for Galphaolf. We found that Ric-8B (for Resistant to Inhibitors of Cholinesterase), a putative GTP exchange factor, is able to interact with Gaolf. Like Gaolf, Ric-8B is predominantly expressed in the mature olfactory sensory neurons and also in a few regions in the brain. The highly restricted and colocalized expression patterns of Ric-8B and Galphaolf strongly indicate that Ric-8B is a functional partner for Galphaolf. We show that Ric-8B is able to potentiate Galphaolf-dependent cAMP accumulation in human embryonic kidney 293 cells and therefore may be an important component for odorant signal transduction. Finally, we show that Ric-8B promotes efficient heterologous expression of ORs. Our results show that Ric-8B enhances accumulation of Galphaolf at the cell cortex, indicating that it promotes functional OR expression probably by improving the efficiency of OR coupling to Galphaolf. Our results demonstrate that the employment of Ric-8B in a high-throughput system will allow the functional screening of the OR family members and thereby provide further insight into the mechanisms of odor perception.

Este trabalho descreve uma simulação computacional biologicamente plausível do sistema olfativo de vertebrados. O modelo construído foi capaz de reproduzir satisfatoriamente características importantes observadas no sistema olfativo de vertebrados, incluindo a recepção de diferentes concentrações e tipos de odores no epitélio olfativo e a propagação dessa informação para o bulbo. Ele também tornou possível a observação de diferentes padrões de resposta odorífera, tanto no epitélio como no bulbo, associados a diferentes odores usados nas simulações. / This work describes a biologically plausible simulation of the olfactory system of vertebrates. The constructed model was capable of reproducing satisfactorily important characterisitics observed in the vertebrate olfactory system, including the reception of different concentrations and odor types at the epithelium and the propagation of this information to the olfactory bulb. Also, it made possible the observation of the different response patterns, both in the epithelium as in the olfactory bulb, associated with different odors used in the simulations.

Esse trabalho consiste no estudo de alguns mecanismos responsáveis pela geração das oscilações elétricas observadas no sistema olfativo de vertebrados e das possíveis funções que essas oscilações possam ter no processamento da informação olfativa. Da-se especial atenção ao papel desempenhado pelo ritmo respiratório e pelas sinapses químicas e elétricas nesse processo. Para realizar essa investigação, foram utilizados modelos computacionais que reproduzem aspectos da anatomia e da fisiologia do epitélio olfativo, do bulbo olfativo e do córtex piriforme. Os modelos foram desenvolvidos e simulados no neurossimulador GENESIS, funcionando no sistema operacional LINUX. A análise dos resultados foi feita no programa MATLAB (Mathworks™). Inicialmente, a tese faz uma descrição do substrato neurobiológico que compõe as camadas iniciais do sistema olfativo, incluindo o epitélio, bulbo e córtex olfativo, e de como a informação olfativa é processada por cada camada, discutindo a importância do sentido olfativo e a relevância da neurociência computacional no estudo da origem e do papel das oscilações elétricas existentes nesse sistema (Capítulo 1). O capítulo 2 descreve os materiais e métodos utilizados para a construção dos modelos computacionais e para análise dos resultados. O capítulo 3 faz uma descrição detalhada do modelo computacional utilizado e dos experimentos realizados com o modelo. Finalmente, o capítulo 4 apresenta e discute os resultados das simulações realizadas e o capítulo 5 estende essa discussão, concluindo a tese. O capítulo 6 contém as referências bibliográficas utilizadas no trabalho. Os resultados do trabalho sugerem que as oscilações elétricas no sistema olfativo poderiam ser geradas em várias estruturas e níveis de organização, abrangendo os níveis moleculares, celulares e de sistemas neurais. E que as sinapses químicas e elétricas, assim como os ritmos respiratórios, podem ter um papel fundamental na geração dessas oscilações. Assim, o modelo construído propõe uma explicação plausível para a origem das oscilações elétricas no sistema olfativo de vertebrados e discute as possíveis funções que essas oscilações teriam no contexto do processamento da informação sensorial. / This work is a study of some mechanisms associated with the generation of electric oscillations in the vertebrate olfactory system. Special attention is given for the role of the respiratory rhythm, chemical synapses and electrical synapses in this process. The possible functions of the electric oscillations in olfactory information processing are explored. A computational model that reproduces aspects of the anatomy and physiology of the olfactory epithelium, bulb and piriform cortex was utilized to realize this investigation. The models were developed and simulated in the GENESIS neurosimulator, running under the LINUX operational system. The analysis of the results was made in the software MATLAB (Mathworks™). In the beginning, the thesis describe the neurobiological substracts of the initial layers of the olfactory system, including the olfactory epithelium, bulb and piriform cortex, and explore how the olfactory information is processed by each layer. The chapter 1 presents the importance of the olfactory sense and the use of computational neuroscience to study the role of the electric oscillations in this system. The chapter 2 explains the material and methods utilized to develop the computational model and to analyse the data generated by the model. The chapter 3 describes the used computational model and the experiments realized with the model. Finally, the chapter 4 presents and discusses the results of the simulations. The chapter 5 extends the discussion and concludes the thesis. The chapter 6 contains the bibliographic references. The results of the work suggest that electric oscillations in the olfactory system could be generated in several structures and organizational levels, including the molecular level, the cellular and neural systems level. In particular, the results shown that chemical and electric synapses, as well as the respiratory rhythm, may have a fundamental role in the generation of these oscillations. Indeed, the constructed model proposes a plausible explanation for the origin of the electrical oscillations in the vertebrate olfactory system and discusses the possible function of these oscillations in the context of sensorial information processing.

The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. Olfactory ensheathing cells (OECs), specialized glial cells within the olfactory nerve, are thought to play an important role in the ongoing growth and replenishment of sensory connections in this system. To gain insight into novel molecules that could mediate OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments revealed very high expression of the fibulin-3 glycoprotein in OECs. To date, research on fibulin-3 has been limited and mainly focused on its involvement in Doyne honeycomb retinal dystrophy, vasculogenesis and tumor formation. As the extracellular matrix associated with OECs is thought to be an important contributor to a growth-permissive environment, the main aim of this thesis was to define a putative role for fibulin-3 during olfactory receptor neuron replacement and regeneration. This hypothesis was investigated in a series of in vitro and in vivo experiments that involved lentiviral vectors to manipulate fibulin-3 gene expression in OECs as well as the use of knock-out mice. Using genetically-modified OECs, experimental data showed that increased levels of fibulin-3 induced morphological changes in OECs and also impeded their migration. Lentiviral vector-mediated expression of fibulin-3 in OECs also had an inhibitory effect on neurite outgrowth from dorsal root ganglion explants. On the other hand, knock-down of fibulin-3 levels via siRNA technology resulted in reduced proliferation. Comparative lesioning experiments in fibulin-3 knock-out and wild-type mice allowed for further assessment of a role for fibulin-3 in olfactory nerve repair in vivo. Two experimental injury models, i.e. epithelial (Triton-X) lesioning and olfactory bulbectomy, were employed. The results obtained were in line with in vitro observations. A lack of fibulin-3 in knock-out mice resulted in a seemingly augmented regeneration of the olfactory epithelium at 10 days post-injury. However, at the latest recovery time point of 42 days post-injury, an impaired recovery of the olfactory epithelium from the experimental insults was observed. Although the precise mechanism for the latter phenomenon is not yet fully understood, our data point towards several factors which include vascular abnormalities and altered cell proliferation within the olfactory epithelium. Additionally, the precise protein distribution of another wide-spread family of extracellular matrix molecules, the laminins, was investigated in this thesis. It was of interest to investigate the spatiotemporal expression of laminin isoforms during iii olfactory nerve development and regeneration as these molecules may have distinct roles in promoting olfactory sensory neuron growth and patterning. In situ hybridization and immunohistochemical studies concluded that laminin-211 and laminin-411 were the most likely candidates to play such a role. In summary, this thesis provides new insights into the role of the extracellular matrix, fibulin-3 in particular, in regulating cell migration, division and axonal growth in the primary olfactory pathway. Such knowledge also gives a greater understanding of the molecular mechanisms by which OEC transplants may enhance axonal regeneration elsewhere in the CNS.

Adult neurogenesis of olfactory receptor neurons (ORNs) occurs in diverse organisms including in decapod crustaceans. This dissertation describes the molecular, cellular, and physiological changes that occur during adult neurogenesis of ORNs in the antennular lateral flagellum (LF) of the spiny lobster Panulirus argus. Examination of the role of splash (spiny lobster achaete scute homolog) in adult neurogenesis and regeneration using in situ hybridization showed splash was not closely associated with the formation of sensory neurons under normal physiological conditions. Damage to the LF, which induces regeneration, enhanced splash expression, suggesting an association between splash with regeneration and repair. This study suggests that splash plays multiple roles in the olfactory organ of adult spiny lobsters. Examination of extracellular and intracellular Ca2+ in mediating spontaneous and odor-induced responses of ORNs, using calcium imaging showed that odor-induced Ca2+ transient responses and spontaneous Ca2+ oscillations in ORN somata are primarily mediated by an influx of extracellular Ca2+ through Co2+ -sensitive Ca2+ channels, but that intracellular Ca2+stores also have some contribution. These responses are independent of TTX-sensitive Na+ channels, suggesting that these Ca2+ responses may reflect receptor potentials. Examination of changes in odor specificity, sensitivity, and temporal responses in adult-born ORNs showed an increase in the percentage of odorant-responsive ORNs as they age from newly-born cells to mature, and a decrease in odorant-responsive ORNs as they senesce. As adult-born ORNs age, there was a decrease in the percentage of ORNs that undergo spontaneous Ca2+ oscillations and an increase in the amplitude of oscillation. ORNs became more broadly tuned as they senesce, and their response profile, defined by the most effective odorant, changed. Odor sensitivity changed with age. This study demonstrated that the physiological response properties of adult-born ORNs changed with functional maturation. Taken together, this dissertation reveals molecular, cellular and physiological changes in adult born ORNs and elucidates mechanisms of adult neurogenesis.

<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>

Our senses are our mind's window to the outside world and determine how we perceive our environment.Sensory systems are complex multi-level systems that have to solve a multitude of tasks that allow us to understand our surroundings.However, questions on various levels and scales remain to be answered ranging from low-level neural responses to behavioral functions on the highest level.Modeling can connect different scales and contribute towards tackling these questions by giving insights into perceptual processes and interactions between processing stages.In this thesis, numerical simulations of spiking neural networks are used to deal with two essential functions that sensory systems have to solve: pattern recognition and prediction.The focus of this thesis lies on the question as to how neural network connectivity can be used in order to achieve these crucial functions.The guiding ideas of the models presented here are grounded in the probabilistic interpretation of neural signals, Hebbian learning principles and connectionist ideas.The main results are divided into four parts.The first part deals with the problem of pattern recognition in a multi-layer network inspired by the early mammalian olfactory system with biophysically detailed neural components.Learning based on Hebbian-Bayesian principles is used to organize the connectivity between and within areas and is demonstrated in behaviorally relevant tasks.Besides recognition of artificial odor patterns, phenomena like concentration invariance, noise robustness, pattern completion and pattern rivalry are investigated.It is demonstrated that learned recurrent cortical connections play a crucial role in achieving pattern recognition and completion.The second part is concerned with the prediction of moving stimuli in the visual system.The problem of motion-extrapolation is studied using different recurrent connectivity patterns.The main result shows that connectivity patterns taking the tuning properties of cells into account can be advantageous for solving the motion-extrapolation problem.The third part focuses on the predictive or anticipatory response to an approaching stimulus.Inspired by experimental observations, particle filtering and spiking neural network frameworks are used to address the question as to how stimulus information is transported within a motion sensitive network.In particular, the question if speed information is required to build up a trajectory dependent anticipatory response is studied by comparing different network connectivities.Our results suggest that in order to achieve a dependency of the anticipatory response to the trajectory length, a connectivity that uses both position and speed information seems necessary.The fourth part combines the self-organization ideas from the first part with motion perception as studied in the second and third parts.There, the learning principles used in the olfactory system model are applied to the problem of motion anticipation in visual perception.Similarly to the third part, different connectivities are studied with respect to their contribution to anticipate an approaching stimulus.The contribution of this thesis lies in the development and simulation of large-scale computational models of spiking neural networks solving prediction and pattern recognition tasks in biophysically plausible frameworks. / <p>QC 20150504</p>