That odors play a significant role in subhuman intraspecies communication is a generally accepted fact explained in part by the production and reception of species specific pheromones. Recently the effects of human produced odors on human communication have received research attention, particularly in the communication of such biological phenomena as menstruation onset and gender assignment. Again pheromones have been posited as the explanatory mechanism. Whether a pheromone-like odor cued process exists in the communication of human emotions is unknown, though a number of anecdotal accounts of odor-emotion interactions suggest that such may be the case. It was the purpose of the present study to investigate a possible odor-emotion interaction by determining whether humans could differentially detect other humans' odor collected under varying emotional conditions.

The objective of this study was to investigate the role of organic cation transporters (OCTs) in the uptake of hydrophilic drugs into the olfactory bulb and subsequently to the brain. Two OCT2 substrates, amantadine and cimetidine were used as model drugs for this purpose. Bovine nasal explants (olfactory and respiratory tissue) were used as an in vitro model for preliminary screening to identify the role of transporters involved in the uptake of drug across these tissues. It was observed from both PCR and immunohistochemistry that OCTs, OCT2, OCTN1 and OCTN2 were present in the bovine respiratory and olfactory mucosa. Transport studies of amantadine in the presence and absence of OCT2 and OCTN2 inhibitors indicated that both these transporters play a role in the transport of amantadine across the bovine respiratory mucosa, whereas transport across the olfactory mucosa was predominantly via OCT2. This was followed by in vivo studies in rats where the blood, striatum and olfactory bulb concentrations of amantadine were determined following intranasal and intra-arterial administration. Shortly after nasal administration, the olfactory bulb concentrations exceeded the concentrations in the striatum suggesting the olfactory pathway to be the major route of uptake. Co-administration of the drug with an OCT2 inhibitor intranasally showed statistically significant reductions in the brain uptake of amantadine. A synergistic inhibitory effect on amantadine uptake was observed with the combined inhibition OCT2 and OCTN2. Additionally, the CNS exposure of these drugs following intranasal administration in the presence and absence of the OCT inhibitors was evaluated using the ratio of the free drug concentrations in the brain compared to plasma. While the plasma concentration profiles were similar both in the presence and absence of inhibition, the free drug ratios were highest when no inhibitor was included. Additionally similiar in vivo studies were also carried out for a second model drug, cimetidine, where cimetidine uptake into the rat brain was found to be significantly reduced in the presence of the OCT2 inhibitor, pentamidine. This demonstrates that there was a greater CNS exposure to each drug when OCT transporters were active, confirming their role in their direct CNS distribution from the nasal cavity to the brain. The results of this study suggest that OCT substrates might be good candidates for the delivery to the brain via the olfactory route.

Chez les mammifères, le système olfactif principal est fonctionnel dans le dernier tiers du stade gestationnel. Le fœtus est donc capable de détecter avec sensibilité, discriminer, et mémoriser les odorants présents dans le liquide amniotique dans lequel il baigne. Chez le rongeur, ces odorants mémorisés in utero sont fondamentaux pour la survie du nouveau-né à la naissance en lui permettant de s’orienter vers la mamelle et de déclencher la tétée. Par la suite, ces odorants vont guider le nouveau-né dans ses choix olfactifs et alimentaires et favoriser ses apprentissages ultérieurs. Or, on sait que l’alimentation de la mère fait varier les clés olfactives des fluides biologiques (liquide amniotique, lait) et donc modifie la nature des sources olfactives rencontrées par le fœtus, puis le nouveau-né dans la période périnatale. Dans le but d’évaluer l’impact d’une exposition odorante périnatale sur le fonctionnement et la maturation du système olfactif en relation avec le développement de préférences médiées par l’olfaction et la modulation du stress au moment du sevrage, nous avons mis en place un modèle d’exposition odorante périnatale via l’alimentation maternelle dans une lignée transgénique murine exprimant le récepteur olfactif I7 couplé à une protéine fluorescente, dont l’odorant préférentiel est l’heptanal. Après validation de la présence d’heptanal dans le liquide amniotique et le lait de ces souris par GC-MS-MS, nos résultats montrent que l’exposition périnatale à l’heptanal entraine une augmentation significative du nombre de glomérules I7 présents au niveau du bulbe olfactif à PN12 par rapport aux animaux contrôles sans modification notable de l’homéostasie tissulaire ou de la transcription du gène I7 au niveau de la muqueuse olfactive. Ce changement structural important au niveau des projections axonales du bulbe olfactif s’accompagne d’un faible effet sur le comportement olfactif des souriceaux dont les capacités de discrimination ne sont que très faiblement améliorées par rapport aux souriceaux non exposés. A PN21, l’effet de l’exposition odorante sur le nombre de glomérules I7 est atténué, mais reste significatif. A ce stade, les souriceaux odorisés montrent une attraction olfactive pour l’heptanal par rapport au groupe contrôle non exposé, bien que l’effet sur un choix entre aliment odorisé ou pas soit plus contrasté. D’un point de vue moléculaire, ces effets sont associés à une diminution de l’expression du récepteur I7, et de gènes de la signalisation neuronale pouvant traduire une modification de la dynamique cellulaire. Et comme le montrent les résultats obtenus en électro-olfactogramme à cet âge, la sensibilité de la muqueuse olfactive vis-à-vis de l’heptanal est diminuée alors qu’elle ne l’est pas pour d’autres composés odorants. Ces travaux montrent que l’exposition périnatale à un odorant s’accompagne d’effets précoces sur le système olfactif qui impactent son organisation et d’effets sur les choix olfactifs qui se renforcent au cours du développement. Enfin, l’effet d’une exposition périnatale à l’heptanal sur la réponse à des conditions stressantes après séparation maternelle au moment du sevrage a été évaluée en enregistrant des souriceaux CD1 élevés dans les mêmes conditions d’odorisation que les souriceaux mI7-GFP adoptés, en open-field odorisé ou non. Nous avons montré que la réaction de stress des souriceaux à PN21, exposés en périnatal à l’heptanal, dans l’open field, est atténuée lorsque l’heptanal est présent dans l’environnement, comparé à la réaction des souriceaux non exposés qui affichent un comportement d’anxiété. Dans l’ensemble, cette étude a donc permis de mettre en évidence les conséquences en terme d’effets comportementaux (préférences médiées par l’olfaction et diminution du stress), structuraux et moléculaires, d’une exposition odorante périnatale chez le jeune, à l’aide d’une lignée transgénique pour laquelle nous disposions de peu de données. / In mammals, the main olfactory system displays all the structural and functional characteristics at the last third of the gestational stage. Thus, the fetus is able to detect, to discriminate, but also to memorize the odorants present in the amniotic fluid in which it bathes. In the rodent, these odors memorized in utero are fundamental for the survival of the newborn at birth by allowing it to trend itself towards food sources. Afterwards, odorants are guiding the newborn to olfactory and food choices and are promoting subsequent learning. It is known that the mother's diet varies the olfactory keys of the biological fluids (amniotic fluid, milk) and thus modifies the nature of the olfactory sources encountered by the fetus and then the newborn in the perinatal period. The neuroanatomic and functional consequences of this impregnation are the subject of recent studies. The objective of this thesis was to characterize the effects of perinatal odor exposure on the maturation and functioning of the olfactory system in relation to the development of olfactory preferences and to a stressing challenge at weaning. For that, we set up a model of perinatal odor exposure through maternal feeding in the mI7-GFP murine transgenic strain expressing the olfactory receptor I7 coupled with the Green Fluorescent Protein (GFP). We evaluated the neuroanatomic, molecular and behavioral consequences on the pups before weaning, and their evolution over time by focusing our efforts on the postnatal stages 3 (PND3), PND12-14 and PND21. Due to the fragility of the transgenic strain, we worked on mI7-GFP mice pups crossfostered by CD1 mice mothers raised under the same conditions after having validated the presence of heptanal in the amniotic fluid of mI7-GFP mice and the milk of CD1 mice by GC-MSMS. We characterized the effects of perinatal odor exposure on the maturation and functioning of the olfactory system in relation to the olfactory preference until weaning (mI7-GFP mice pups under CD1 adoptive mothers) and on the stress reaction to maternal separation at weaning (CD1 mice pups under biological mother). Our results show that perinatal exposure to heptanal leads to a significant increase in the number of I7 glomeruli in the olfactory bulb (OB) associated to a slight modification of the tissue homeostasis in the olfactory mucosa (OM) and to subtle differencies in heptanal sensitivity and preferences, that are amplified at PND21. From a molecular point of view, these effects are associated to a down-regulation of the expression of the I7 receptor and genes of neuronal signaling and an odorantspecific decrease in EOG response which may highlight a modification of the cellular dynamics. Finally, the effect of perinatal exposure to heptanal on the response to stressing conditions after maternal separation was assessed by recording CD1 mice pups grown under the same conditions than fostered mi7 mice on odorized and non odorized open-field. We showed that the reaction of odorized mice in the open field at PND21 is attenuated when the odorant is present in the environment, compared to non odorized mice that display anxiety-like behavior. Overall, this study demonstrates the consequences of a perinatal odorant exposure in the young, in terms of behavior (olfactory preference and anxiety), structural and molecular plasticity of the olfactory system, on a transgenic strain for which we had little available data.

The olfactory system is of great interest in research due to the olfactory epithelium’s regenerative capability and as a potential as a source of neural stem cells. The olfactory sensory neurons are constantly being replaced by the stem cells that lie at the base of the olfactory epithelium. These stem cells also remain intact after an injury to the epithelium and lead to the regeneration of the olfactory epithelium. We have developed a fate mapping technique to trace axonal regrowth from newly born olfactory sensory neurons using an inducible Cre-ERT2 model after chemical ablation by the drug methimazole. Our data shows that newly generated olfactory sensory neurons labeled 1 day after chemical ablation by injection of 4-HO-tamoxifen extend an axon that reaches the olfactory bulb and extend to the glomeruli in a timeline that is consistent with control mice that received 4-HO-tamoxifen but were injected with saline 1 day prior. In addition, we assessed the functional recovery of the olfactory epithelium by testing the ability of mice to find a hidden cookie after methimazole injection. Mice were tested at 3 and 14 days post methimazole. There was a severe impairment in the ability to find a hidden cookie at 3 days post methimazole. The mice tested at 14 days post methimazole showed an improvement in the ability to find the cookie but the latency to find the cookie was still significantly higher than controls. In conclusion, while we demonstrate that axons extend to the olfactory bulb and the glomeruli earlier than 14 days, our behavioral data suggest that there must be a critical number of axons that must reach each specific glomerulus to regain function of the olfactory system.

Neurons in the olfactory epithelium that are responsible for detecting the odors we smell are constantly dying. However, the olfactory system has the unique ability to regenerate new neurons in order for the sense of smell to be maintained. After a new sensory neuron is born in the olfactory epithelium, it must extend a new axon that will travel to the olfactory bulb and make specific synaptic contact so that the odor information from the epithelium can be coded and sent to the higher cortical areas of the brain. The olfactory system’s ability to recover is also even more complex in that it is capable of regeneration after an injury in which a portion or even the entire olfactory epithelium is removed. A well established model for this type of injury in the olfactory epithelium is by inducing a chemical ablation by injection of the drug methimazole. A specific interest in the regenerative process after injury is the mechanism by which axonal debris from the dead neurons is removed. After ablation of the olfactory epithelium, the cell bodies of the neurons detach but their axons remain intact. The axonal debris must not only be removed, but must also be done so in a way that minimizes inflammation in order for new axons to be able to extend to the olfactory bulb. Axonal debris removal has been characterized both in vitro and during development. However, the mechanism of debris removal has yet to be characterized after an injury. Our lab has studied different engulfment proteins in the olfactory bulb after injury using RT-qPCR and found specific temporal expression profiles at 3, 14 and 21 days post injury. Our initial investigations involved some known engulfment proteins such as Jedi1, GULP, and Megf10. However, we found that these proteins are downregulated after an injury. Further investigation has shown that the proteins Cd11b and TLR2 are upregulated after injury. These changes in expression can begin to shed light on the mechanism of axonal debris removal after an injury and can further be used to study how inflammation is suppressed in order to allow for axon extension and synaptic contact to be reestablished.

The initial step in odor recognition by the nose is the binding of odorant molecules to receptor sites embedded in the dendritic membranes of olfactory receptor cells. Despite considerable interest and experimentation into the nature of these receptor sites, little is known of their specificity to different types of odorant molecules. This lack of knowledge partially stems from the fact that the nature of receptor proteins is most effectively studied when specific and irreversible inhibitors are available for use as chemical probes, yet no such agents have been discovered for use in the olfactory system. A series of alkylating agents and other chemically active odorants were tested to determine whether they might react with specific odorant receptors and modify olfactory responses. Electroolfactogram (EOG) recordings were obtained before, during, and after treatment of the olfactory mucosae of grass frogs (Rana pipiens) with a chemically active odorant.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22787号 / 農博第2430号 / 新制||農||1081(附属図書館) / 学位論文||R2||N5307(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 井鷺 裕司, 教授 森 直樹, 准教授 大澤 直哉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Olfactory sensory neurons (OSNs) are contained within the olfactory epithelium (OE) and are responsible for detecting odorant molecules in the air. The exposure of OSNs to the external environment is necessary for their function, but it also leaves them exposed to potentially harmful elements and thus results in a high turnover rate. Despite the high turnover, the olfactory sense is maintained throughout life through the division of a population of stem cells that produce new OSNs both during normal turnover and after an injury occurs in the OE. When new OSNs are born, they must extend axons from the OE to the olfactory bulb (OB) where they make specific synaptic contacts. To determine the timeline of axon extension in normal turnover and after a methimazole-induced injury, we used fate-tracing utilizing an inducible Cre-LoxP model in which a fluorescent reporter was expressed by neuronal precursors and subsequently used to track axonal growth as the OSNs matured. Our results show that axon extension in both conditions follow the same timeline. However, markers of synaptic connectivity in the OB were delayed after injury. The delay in synaptic connectivity was also corroborated with delays in olfactory behavior after injury, which took 40 days to recover to control levels. Additionally, we investigated the process of removal of axonal debris created after an injury. Immunohistochemical analysis after injury indicated upregulation of IBA1+ cells within the 3 olfactory nerve layer of the OB, suggesting a role of microglia in this process. These microglia also showed an activated morphology and some had very large cell bodies with multiple nuclei. Furthermore, qPCR analysis of post-injury OB tissue shows upregulation of the CD11b receptor that is expressed on microglia. Our results have also shown upregulation of components of the complement pathway after injury, which is suggestive of a mechanism that underlies axonal debris removal after injury in the OB. Taken together, these results shed light on the process by which the olfactory system is able to recover after injury and could lead to discovery of mechanisms that could translate to treatments for injuries in other areas of the nervous system.

Les interactions entre individus sont un socle vital pour l’organisation des colonies d’abeilles, en particulier lors du recrutement pour le butinage. Outre la communication de la localisation d’une source de nourriture par la fameuse danse, les abeilles recrutées apprennent les caractéristiques de l’odeur des fleurs butinées au cours de transferts de nectar (trophallaxie). Les mécanismes de cet apprentissage ne sont pas encore éclaircis car il est parfois effectif sans aucun transfert de nectar, suggérant que d’autres mécanismes, comme par exemple d’apprentissage social, sont impliqués. Nous avons reproduit cette interaction en laboratoire, suivant un protocole basé sur le conditionnement olfactif appétitif de la réponse d’extension du proboscis (REP). Ici, un composé odorant initialement neutre (Stimulus conditionnel) était associé à un contact avec une congénère (Stimulus Inconditionnel social), sans récompense sucrée. Nos expériences montrent que ce simple contact social entre congénères peut constituer un renforcement pour les abeilles. A la suite de cette association, celles-ci montrent donc des REP à l’odeur préalablement associée au contact social. Nos expériences montrent de plus que des contacts antennaires entre les abeilles sont indispensables à l’efficacité de cet apprentissage social, représentant un indice social tactile. Nous avons alors développé un dispositif permettant d’enregistrer les mouvements des antennes de manière précise et à haute vitesse (90 hz) et étudié les différents facteurs modulant les mouvements antennaires des abeilles. Les abeilles montrent des réponses contrastées et reproductibles à des odeurs de valeurs biologiques différentes. De plus, le couplage de ces enregistrements à des expériences de conditionnement associatif montre que ces réponses antennaires sont plastiques et modifiées par l’expérience des individus. Ce travail a permis de mettre en lumière un nouveau type d’apprentissage social chez les insectes et d’approfondir l’étude des mouvements antennaires comme indicateurs de l’état motivationnel, attentionnel et physiologique des abeilles ainsi que de la valence des stimuli perçus. / In honeybees, interactions between individuals are cornerstones for the organization of the colony, especially during recruitment for foraging. Besides learning the location of a food source thanks to the well-known dance, the recruited bees learn the characteristics of odors of foraged flowers through nectar transfer (trophallaxis). The underlying mechanisms are still unclear because this learning can occur without any nectar transfer, suggesting that other, probably social, learning mechanisms are involved. We reproduced this interaction in the lab, using a protocol based on the appetitive olfactory conditioning of the proboscis extension response (PER). Here, an initially neutral odorant (conditioned stimulus) was associated with a contact with a nestmate (social unconditioned stimulus), without any sugar reward. Our experiments show that this simple social contact between workers can act as a reinforcement for bees. As a result, they show PER to the odor previously associated with a social contact. We further demonstrate that antenna contacts are essential for the effectiveness of this social learning, representing a tactile social cue. We thus developed a system allowing to record bees’ antennal movements accurately and at high frequency (90 hz). We then determined the factors modulating bees’ antennal movements. First, we show that bees display contrasted and reproducible responses to odors of different biological values. Second, the coupling of these recordings with associative conditioning experiments shows that these antennal responses are plastic and modified by individual experience. This work has shed light on a new type of social learning in insects and has furthered our understanding of antennal movements as indicators of the motivational, attentional and physiological state of bees and of the valence of perceived stimuli.

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