Dissertation (Ph.D.)--Auburn University, / Abstract. Vita. Includes bibliographic references.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Weissburg, Marc; Committee Member: Hay, Mark; Committee Member: Kubanek, Julia; Committee Member: Webster, Donald; Committee Member: Yen, Jeannette. Part of the SMARTech Electronic Thesis and Dissertation Collection.

The cerebral cortex continuously encodes new sensory information and organizes it within an experiential intracortical framework. The cortical integration of internal and external information forms the associations that are the basis for higher order sensory representation, and ultimately, perception. Deciphering the cellular and synaptic principles of sensory-cortical integration requires a system with a simplified interface between the internal and external worlds. The piriform cortex provides a relatively simple substrate for the study of intracortical modulation of sensory coding. Within piriform, primary sensory information from the olfactory bulb converges onto neurons in a single cortical layer, where it directly integrates with intracortical input. The major barrier to studying intracortical influences on sensory representation in piriform has been the inability to isolate single types of intracortical input. Here, we use optogenetic techniques to functionally isolate two important classes of intracortical input to piriform pyramidal neurons, and slice electrophysiology to assess their synaptic properties. We first expressed channelrhodopsin in a small subset of piriform neurons, effectively isolating the recurrent synapses formed onto piriform pyramidal neurons by their peers. Recurrent collaterals form strong excitatory connections that extend throughout piriform without spatial attenuation in strength, linking distant piriform neurons. This extensive recurrent network is constrained by powerful disynaptic inhibition, which can also reduce activation by primary sensory inputs in a timing-dependent manner. Next, we functionally isolated inputs to the piriform from the anterior olfactory nucleus (AON), an early target of olfactory bulb output whose role in olfaction is largely unknown. The AON makes weaker excitatory connections with piriform, but unlike recurrent connections, these inputs do not drive strong disynaptic inhibition. Sequential activation of AON inputs leads to pronounced summation that boosts piriform activation in an NMDA-receptor-dependent manner, and may enhance plasticity of AON-to-piriform synapses. The AON is a potentially powerful modulator of piriform cortex, whose role in odor information processing merits further study. Our results collectively illustrate critical features of intracortical input classes to piriform cortex, and how these inputs may have distinct roles in shaping odor representations and olfactory learning.

The mushroom body (MB) is the site of odor association learning in Drosophila.  In the canonical model, there are two types of reinforcing dopamine neurons (DANs): one set for rewarding unconditioned stimuli (US), and one responding to aversive US.  When DANs are activated together with an odor (the conditioned stimulus, or CS), plasticity is induced in the downstream output neurons (MBONs).  We have identified a DAN (V1) that surprisingly responds preferentially to odors, and responds weakly or not at all to various classical US.  In order to explore the relationship between V1 odor responses and the established roles of the MB, I characterized the responses of DAN V1, and probed its relationship to odor-driven behavior, associative conditioning, and activity in other MB compartments. These data show that V1 receives recurrent input from identified MBONs, contributes to the activity of an MBON that enhances alerting behavior, and that its odor responses are modulated by conditioning. We therefore present the study of the alpha2 compartment, which V1 innervates, as the dissection of an atypical compartment of the MB, one that acts as a hub by which various information from other compartments and brain areas is integrated in order to alter a behavioral response to odor. This work furthers our understanding of the MB not simply as an engine of classical learning, but as a system of diverse interconnected modules that allow coordinated fine control of behavior.

The mammalian olfactory system is capable of decoding complex mixtures of volatile chemical odorants into identifiable percepts. While the general mode of peripheral signal transduction is largely known, the mechanism relies on a rather complicated combinatorial “olfactory code”, where each of the hundreds of expressed odorant receptors (ORs) detects multiple odorants, and a given odorant in turn activates multiple ORs (Malnic, Hirono et al. 1999). Since the first identification of mammalian ORs in 1991, the deorphanization, i.e. solving of the substrate, of ORs has proven to be a challenge. Many attempts at systematic monitoring of the olfactory code have seen marginal successes for a number of reasons. First of all, there are still no solved structures of mammalian ORs to be used for high throughput computational modeling. Second, experimental validation methods such as heterologous expression still face considerable challenges. Lastly, primary chemical features of odors that allow for OR tuning are not yet defined. The traditional organic chemistry-based classification of odorants fails to predict biological activity, while percept-based computational analyses isolate esoteric descriptors that are difficult to chemically manipulate. Receptor level structure-activity analysis can provide a missing context to the odorant discrimination in the peripheral olfactory system. A critical finding by Manic et al (1999) indicates that each mature olfactory sensory neuron (OSN) only expresses one type of OR, allowing for high throughput screening of carefully crafted odorant panels using dissociated OSN calcium imaging. A few bioisosteric substitutions widely utilized in medicinal chemistry were used to construct odorant panels, showing greater success in defining odorant-OR interaction than previously used organic chemistry-based clustering methods. Among classical substitutions used by medicinal chemists, heteroaromatic ring exchanges are especially well tolerated when heteroatoms with a similar topological polar surface area (TPSA) are used as replacements. Among odorants with differing TPSA, it is likely that an OR activated by analogous odorants at two extremes of the TPSA spectrum will be activated by an odorant with an intermediate TPSA. Flipping of a polar functional group, which is often used with amides in drug target replacements, is well tolerated by the ORs in esters. Furthermore, there is a predictable activation pattern relative to number of carbons in a hydrophobic chain uninterrupted by polar epitopes. Using binary mixtures, the OR activity can be further surveyed through enhancement or inhibition of OSN activation signals. Odorants activating a smaller subset of an OR population may also be binding to a larger subset of ORs, resulting in mixture inhibition. Specifically, this work indicates that extracted odorant fragments may be binding but not activating some of the OR repertoire of the original odorant. The concept of non-classical bioisosteres is applied to the OR repertoire using aliphatic and aromatic aldehydes. It appears that the specialized electronics of a fully conjugated benzene ring can in fact be dispensable, only acting as conformational restrictor of the odorant in most cases. Not only do analogous non-conjugated systems substitute well for benzaldehyde, but so do non-cyclic odorants possessing tiglic moieties. Conformationally restricted extractions act as more faithful replacements for larger molecules in a subset of ORs. While the dissociated OSN results alone have broad implications for binding patterns of GPCRs in general, simple behavioral tests in mice using the same odorant panels indicate concrete perceptual links to medicinal chemistry-based odorant discrimination. The results from the behavioral data suggest that there may be a maximum constraint for percent OSN activation for two sequentially presented odors to be interpreted as the “same”. The results open a window to exploring other medicinal chemistry-based substitutions. Furthermore, many methodological improvements have been made over the past decade to allow for increased efficiency of deorphanization and validation of ORs.

The evolution of genes and genomes has attracted great interest. The research presented here is an examination of genomes at three distinct levels, protein evolution, gene family evolution, and TE content regulation. First at a genetic level, I conducted an analysis of the salivary androgen-binding proteins (ABPs). I focused on comparing patterns of molecular evolution between the Abpa gene expressed in the submaxillary glands of species of New World and Old World muroids and found that in both sets of rodents, the Abpa gene expressed in the submaxillary glands appear to be evolving under sexual selection, suggesting ABP might play a similar biological role in both systems. Thus, ABP could be involved with mate recognition and species isolation in New World as well as Old World muroids. Second I examined the largest gene family in vertebrate olfactory receptors (ORs) among birds and reptiles. I found that the number of intact OR genes in sauropsid genomes analyzed ranged over an order of magnitude, from 108 in the lizard to over 1000 in turtles. My results suggest that different sauropsid lineages have highly divergent OR repertoire compositions. These differences suggest that varying rates of gene birth and death, together with selection related to diverse natural histories, have shaped the unique OR repertoires observed across sauropsid lineages. Lastly, I studied the interactions between transposable elements (TEs) and PIWI-interacting RNAs (piRNAs) among laurasiatherian mammals. piRNAs are predominantly expressed in germlines and reduce TE expression and risks associated with their mobilization. I found that within TE types, families that are the most highly transcribed appear to elicit the strongest ping-pong response. This was most evident among LINEs, but the relationships between expression and PPE was more complex among SINEs. I also found that the abundance of insertions within piRNAs clusters strongly correlated with genome insertions and there was little evidence to suggest that piRNA clusters regulated TE silencing. In summary, the piRNA response is efficient at protecting the genome against TE mobility, particularly LINEs, and can have an evolutionary impact on the TE composition of a genome.

For many organisms the sense of smell is critical to survival. Some olfactory stimuli elicit innate responses that are mediated through hardwired circuits that have developed over long periods of evolutionary time. Most olfactory stimuli, however, have no inherent meaning. Instead, meaning must be imposed by learning during the lifetime of an organism. Despite the dominance of olfactory stimuli on animal behavior, the mechanisms by which odorants elicit learned behavioral responses remain poorly understood. All odor-evoked behaviors are initiated by the binding of an odorant to olfactory receptors located on sensory neurons in the nasal epithelium. Olfactory sensory neurons transmit this information to the olfactory bulb via spatially organized axonal projections such that individual odorants evoke a stereotyped map of bulbar activity. A subset of bulbar neurons, the mitral and tufted cells, relay olfactory information to higher brain structures that have been implicated in the generation of innate and learned behavioral responses, including the cortical amygdala and piriform cortex. Anatomical studies have demonstrated that the spatial stereotypy of the olfactory bulb is maintained in projections to the posterolateral cortical amygdala, a structure that is involved in the generation of innate odor-evoked responses. The projections of mitral and tufted cells to piriform cortex however appear to discard the spatial order of the olfactory bulb: each glomerulus sends spatially diffuse, apparently random projections across the entire cortex. This anatomy appears to constrain odor-evoked responses in piriform cortex: electrophysiological and imaging studies demonstrate that individual odorants activate sparse ensembles that are distributed across the extent of cortex, and individual piriform neurons exhibit discontinuous receptive fields such that they respond to structurally and perceptually similar and dissimilar odorants. It is therefore unlikely that olfactory representations in piriform have inherent meaning. Instead, these representations have been proposed to mediate olfactory learning. In accord with this, lesions of posterior piriform cortex prevent the expression of a previously acquired olfactory fear memory and photoactivation of a random ensemble of piriform neurons can become entrained to both appetitive and aversive outcomes. Piriform cortex therefore plays a central role in olfactory fear learning. However, how meaning is imparted on olfactory representations in piriform remains largely unknown. We developed a strategy to manipulate the neural activity of representations of conditioned and unconditioned stimuli in the basolateral amygdala (BLA), a downstream target of piriform cortex that has been implicated in the generation of learned responses. This strategy allowed us to demonstrate that distinct neural ensembles represent an appetitive and an aversive unconditioned stimulus (US) in the BLA. Moreover, the activity of these representations can elicit innate responses as well as direct Pavlovian and instrumental learning. Finally activity of an aversive US representation in the basolateral amygdala is required for learned olfactory and auditory fear responses. These data suggest that both olfactory and auditory stimuli converge on US representations in the BLA to generate learned behavioral responses. Having identified a US representation in the BLA that receives convergent olfactory information to generate learned fear responses, we were then able to step back into the olfactory system and demonstrate that the BLA receives olfactory input via the monosynaptic projection from piriform cortex. These data suggest that aversive meaning is imparted on an olfactory representation in piriform cortex via reinforcement of its projections onto a US representation in the BLA. The work described in this thesis has identified mechanisms by which sensory stimuli generate appropriate behavioral responses. Manipulations of representations of unconditioned stimuli have identified a central role for US representations in the BLA in connecting sensory stimuli to both innate and learned behavioral responses. In addition, these experiments have suggested local mechanisms by which fear learning might be implemented in the BLA. Finally, we have identified a fundamental transformation through which a disordered olfactory representation in piriform cortex acquires meaning. Strikingly this transformation appears to occur within 3 synapses of the periphery. These data, and the techniques we employ, therefore have the potential to significantly impact upon our understanding of the neural origins of motivated behavior.

As células provenientes do epitélio olfatório apresentam capacidade regenerativa durante toda a vida, embora este mecanismo ainda não esteja completamente elucidado. O potencial de diferenciação de células-tronco provenientes do epitélio olfatório de cães sem raça definida (Canis familiaris Linnaeus, 1758) foi avaliado utilizando-se 12 cães adultos e 12 cães com 60 dias de vida intra-uterina, oriundos do Hospital Veterinário da FMVZ-USP. Após coletado, o epitélio olfatório foi submetido a protocolo histológico padrão para hematoxilina-eosina, azul de toluidina e PAS. O material fixado em glutaraldeído 2,5% foi processado para observação sob microscopia eletrônica de transmissão. O índice de proliferação nuclear foi medido para detecção de PCNA e Ki67 nuclear. Células foram expandidas em cultura em meio D-MEM/F-12 acrescido de Soro Fetal Bovino hyclone (CO2-95% à 37ºC) para caracterização e diferenciação celular (osteogênica, adipogência e neurogênica). O cultivo das células provenientes do epitélio olfatório de cães com 60 dias de vida intra-uterina mostrou maior evolução em cultura quando comparados aos animais adultos, sendo as primeiras utilizadas para execução dos protocolos de caracterização e diferenciação. As células em cultivo apresentaram expressão CD29 positiva e marcação positiva para OCT-4, citoqueratina 18 (Ck18) e vimentina. A diferenciação osteogênica demonstrou, ao final de 21 dias, células com morfologia típica, caracterizadas pela coloração de Alizarin Red e Von Kossa. A diferenciação adipogênica mostrou pouco número de células, apresentando grânulos adipogênicos, corados por Oil Red, contudo sem morfologia típica. A diferenciação neurogênica demonstrou células que expressaram marcação para GFAP, Neurofilamentos, Oligodendrócitos e βtubulina III. As células isoladas a partir do epitélio olfatório de cães com 60 dias de vida intra-uterina evidenciaram populações de células-tronco, determinadas pela expressão de marcadores específicos e diferenciação em outros tipos de tecido, devendo-se considerar este epitélio como uma fonte potencial para aquisição de células, particularmente progenitores neuronais, que possam somar aos estudos e seu uso em terapia celular. / Olfactory cells demonstrate regenerative capacity along life, although, its mechanism is still obscure. We evaluated the differentiation capacity of olfactory stem cell of mongrel dogs (Canis familiaris Linnaeus, 1758) in 12 adult dogs and 12 fetuses at term (60 days of pregnancy) from the Veterinary Hospital of FMVZ-USP. Following the sampling, the epithelia were submitted to standard histological process and stained with HE, toluidine blue and PAS. Samples fixed with 2.5% glutaraldehyde solution were processed for transmission electron microscopy. Proliferative index were obtained with the detection of PCNA and Ki67. Cells were kept in culture in D-MEM/F-12 medium supplemented with hyclone bovine fetal serum (CO2-95% at 37ºC) for characterization and cell differentiation (osteogenesis, adipogenesis, and neurogenesis). Cell culture of fetuses at term demonstrated higher evolution in comparison to adult animals, being submitted to the protocols of characterization and differentiation. Cell culture demonstrated positive reaction for CD29, OCT-4, Ck18 (citokeratin) and vimentin. Osteogenic differentiation demonstrated, 21 days, typical morphological cells characterized by Alizarin Red and Von Kossa staining. Adipogenic differentiation demonstrated less number of cells containing granules, stained with Oil Red, although being not typical shape. Neurogenic differentiation demonstrated positive staining for GFAP, Neurofilament, Oligodendrocyte and III β-tubulin. Cells isolated from epithelia of fetuses at term demonstrated population of stem cells determined by the expression of specific-staining and differentiation into other type of cells, which lead us to consider the olfactory epithelia as a source of potential stem cells particularly for neurogenic differentiation to be applied for further studies in cell therapy.

As células provenientes do epitélio olfatório apresentam capacidade regenerativa durante toda a vida, embora este mecanismo ainda não esteja completamente elucidado. O potencial de diferenciação de células-tronco provenientes do epitélio olfatório de cães sem raça definida (Canis familiaris Linnaeus, 1758) foi avaliado utilizando-se 12 cães adultos e 12 cães com 60 dias de vida intra-uterina, oriundos do Hospital Veterinário da FMVZ-USP. Após coletado, o epitélio olfatório foi submetido a protocolo histológico padrão para hematoxilina-eosina, azul de toluidina e PAS. O material fixado em glutaraldeído 2,5% foi processado para observação sob microscopia eletrônica de transmissão. O índice de proliferação nuclear foi medido para detecção de PCNA e Ki67 nuclear. Células foram expandidas em cultura em meio D-MEM/F-12 acrescido de Soro Fetal Bovino hyclone (CO2-95% à 37ºC) para caracterização e diferenciação celular (osteogênica, adipogência e neurogênica). O cultivo das células provenientes do epitélio olfatório de cães com 60 dias de vida intra-uterina mostrou maior evolução em cultura quando comparados aos animais adultos, sendo as primeiras utilizadas para execução dos protocolos de caracterização e diferenciação. As células em cultivo apresentaram expressão CD29 positiva e marcação positiva para OCT-4, citoqueratina 18 (Ck18) e vimentina. A diferenciação osteogênica demonstrou, ao final de 21 dias, células com morfologia típica, caracterizadas pela coloração de Alizarin Red e Von Kossa. A diferenciação adipogênica mostrou pouco número de células, apresentando grânulos adipogênicos, corados por Oil Red, contudo sem morfologia típica. A diferenciação neurogênica demonstrou células que expressaram marcação para GFAP, Neurofilamentos, Oligodendrócitos e βtubulina III. As células isoladas a partir do epitélio olfatório de cães com 60 dias de vida intra-uterina evidenciaram populações de células-tronco, determinadas pela expressão de marcadores específicos e diferenciação em outros tipos de tecido, devendo-se considerar este epitélio como uma fonte potencial para aquisição de células, particularmente progenitores neuronais, que possam somar aos estudos e seu uso em terapia celular. / Olfactory cells demonstrate regenerative capacity along life, although, its mechanism is still obscure. We evaluated the differentiation capacity of olfactory stem cell of mongrel dogs (Canis familiaris Linnaeus, 1758) in 12 adult dogs and 12 fetuses at term (60 days of pregnancy) from the Veterinary Hospital of FMVZ-USP. Following the sampling, the epithelia were submitted to standard histological process and stained with HE, toluidine blue and PAS. Samples fixed with 2.5% glutaraldehyde solution were processed for transmission electron microscopy. Proliferative index were obtained with the detection of PCNA and Ki67. Cells were kept in culture in D-MEM/F-12 medium supplemented with hyclone bovine fetal serum (CO2-95% at 37ºC) for characterization and cell differentiation (osteogenesis, adipogenesis, and neurogenesis). Cell culture of fetuses at term demonstrated higher evolution in comparison to adult animals, being submitted to the protocols of characterization and differentiation. Cell culture demonstrated positive reaction for CD29, OCT-4, Ck18 (citokeratin) and vimentin. Osteogenic differentiation demonstrated, 21 days, typical morphological cells characterized by Alizarin Red and Von Kossa staining. Adipogenic differentiation demonstrated less number of cells containing granules, stained with Oil Red, although being not typical shape. Neurogenic differentiation demonstrated positive staining for GFAP, Neurofilament, Oligodendrocyte and III β-tubulin. Cells isolated from epithelia of fetuses at term demonstrated population of stem cells determined by the expression of specific-staining and differentiation into other type of cells, which lead us to consider the olfactory epithelia as a source of potential stem cells particularly for neurogenic differentiation to be applied for further studies in cell therapy.

Pattern formation during development is guided by tightly controlled gene regulatory networks that lead to reproducible cell fate outcomes. However, stochastic choices are often employed to further diversify cell fates. These two mechanisms are closely interlinked in the mouse olfactory system, where stochastic expression of one of one out of >1,000 olfactory receptor (OR) genes is restricted to anatomical segments, or “zones”, organized along the dorsoventral axis of the olfactory epithelium (OE). Despite recent progress in understanding the processes underlying OR choice, the mechanism by which the dorsoventral position of an olfactory sensory neuron (OSN) dictates its OR repertoire has remained elusive and is the focus of this thesis. To gain insight into a possible mechanism I compared the transcriptomes, chromatin landscape, and nuclear architecture of cells isolated from ventral and dorsal zonal segments of the OE. I determined the developmental window in which cells become restricted in their zonal OR repertoire and found this coincided with both the deposition of heterochromatic histone marks H3K9me3 and H3K79me3 on OR genes and their coalescence into a multi-chromosomal compartment. Comparing heterochromatin levels and OR compartment composition in OSNs from different zones, I determined in each case OR genes with more dorsal indexes have higher levels of H3K9me3/H3K79me3 and thus become silenced, while OR genes with more ventral indexes have no heterochromatin and consequently are excluded from OR compartments. Thus, ORs that are “competent” for activation are relatively more accessible, while still being recruited into the OR compartment where they can interact with the proximally positioned enhancer hub. I found that this mechanism is regulated by Nfi family transcription factors that are expressed in a ventral (high) to dorsal (low) gradient in the OE. Deletion of Nfi A, B and X transforms the heterochromatin and OR compartmentalization in ventral OSNs to a more dorsal state, and shifts their preferred OR repertoire towards more dorsal ORs. This result implicates Nfi proteins as key regulators of zonal OR expression. Finally, I probed the nuclear architecture in single cells to look for the source of stochastic choice within zonal segments. I found high variability in inter-chromosomal OR compartment and enhancer hub composition between individual OSNs that stemmed from the unpredictable and variable positioning of chromosomes in the interphase nucleus. Overall, this thesis provides evidence for a mechanism of zonal OR choice that combines deterministic restrictions imposed by a gradient of Nfi with random inter-chromosomal contacts.