Spike-timing dependent plasticity and connectivity in primate sensorimotor cortex

Thesis (Ph.D.)--University of Washington, 2016-06

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

Scanning Laser Optical Tomography Resolves Structural Plasticity during Regeneration in an Insect Brain

Background: Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system. Methodology: We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx. Conclusions: This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.

Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality

Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56(low) NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56(low) NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94(hi)/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality.

Moringa oleifera plasticity to face climatic changes

Extreme abiotic factors, such drought combined with heat waves and/or high UVB radiation are predicted to become more frequent in the future. The impact on plant production of these challenges on multipurpose Moringa oleifera L. remains unclear. A susceptibility of this species may lead to increase poverty in endangered regions. M. oleifera is a woody species native from sub-Himalaya regions under high climate stress pressure. The interest on this species is emerging due to its several medicinal properties and its nutritional value. Agropharmaceutical industry is interest in this species too. To understand the impact of increased climate factors, young (2 months old) plants of this species were exposed to water deficit (WD) and UVB (alone or combined). WD and WD+UVB imposition consists of unwater for 4 days. After 1 day withholding water, UVB and WD+UVB were irradiated with 26.3 kJ m-2 distributed per 3 days. Immediately after treatment exposition (1 day) and after 10 days, plant water status, growth, carbon metabolism and oxidative stress were measured. Overall no significant differences were observed in WD, regarding the parameters analysed, except on gas exchanges, MDA and phenols. The plants exposed to UVB showed, in general, more severe effects, as higher pigment content, MDA and membrane permeability, while no changes were observed in the total antioxidant activity. Plants exposed to UVB+WD, despite changes observed, the impact was lower than the one observed in UVB exposed plants, meaning that a protective/adaptive mechanism was developed in the plants under combined stressors. On the other hand, in all treatments the net CO2 assimilation rate decreased. Results suggest that M. oleifera has some tolerance to WD and UVB, and that develops mechanism of adaptation to these two types of stress that often arise in combination under a climate change scenario.

Calcium response of KCl-excited populations of ventricular myocytes from the European sea bass (Dicentrarchus labrax): a promising approach to integrate cell-to-cell heterogeneity in studying the cellular basis of fish cardiac performance

Climate change challenges the capacity of fishes to thrive in their habitat. However, through phenotypic diversity, they demonstrate remarkable resilience to deteriorating conditions. In fish populations, inter-individual variation in a number of fitness-determining physiological traits, including cardiac performance, is classically observed. Information about the cellular bases of inter-individual variability in cardiac performance is scarce including the possible contribution of excitation-contraction (EC) coupling. This study aimed at providing insight into EC coupling-related Ca2+ response and thermal plasticity in the European sea bass (Dicentrarchus labrax). A cell population approach was used to lay the methodological basis for identifying the cellular determinants of cardiac performance. Fish were acclimated at 12 and 22 A degrees C and changes in intracellular calcium concentration ([Ca2+](i)) following KCl stimulation were measured using Fura-2, at 12 or 22 A degrees C-test. The increase in [Ca2+](i) resulted primarily from extracellular Ca2+ entry but sarcoplasmic reticulum stores were also shown to be involved. As previously reported in sea bass, a modest effect of adrenaline was observed. Moreover, although the response appeared relatively insensitive to an acute temperature change, a difference in Ca2+ response was observed between 12- and 22 A degrees C-acclimated fish. In particular, a greater increase in [Ca2+](i) at a high level of adrenaline was observed in 22 A degrees C-acclimated fish that may be related to an improved efficiency of adrenaline under these conditions. In conclusion, this method allows a rapid screening of cellular characteristics. It represents a promising tool to identify the cellular determinants of inter-individual variability in fishes' capacity for environmental adaptation.

Plasticity in Insect Olfaction: To Smell or Not to Smell?

International audience

Maternal Copy Number Variants (CNV) transmission to their Autism Spectrum Disorder (ASD) sons correlates with phenotypic traits

Background: Common neurodevelopmental disorder, global prevalence ~1 %; Persistent deficits in social communication and social interaction; restricted and repetitive behavior, interests, or activities; Highly heterogeneous clinical presentation; Male to female ratio ~4:1.

Contrasting effects of historical contingency on phenotypic and genomic trajectories during a two-step evolution experiment with bacteria

Background The impact of historical contingency, i.e. the past evolutionary history of a population, on further adaptation is mostly unknown at both the phenotypic and genomic levels. We addressed this question using a two-step evolution experiment. First, replicate populations of Escherichia coli were propagated in four different environmental conditions for 1000 generations. Then, all replicate populations were transferred and propagated for further 1000 generations to a single new environment. Results Using this two-step experimental evolution strategy, we investigated, at both the phenotypic and genomic levels, whether and how adaptation in the initial historical environments impacted evolutionary trajectories in a new environment. We showed that both the growth rate and fitness of the evolved populations obtained after the second step of evolution were contingent upon past evolutionary history. In contrast however, the genes that were modified during the second step of evolution were independent from the previous history of the populations. Conclusions Our work suggests that historical contingency affects phenotypic adaptation to a new environment. This was however not reflected at the genomic level implying complex relationships between environmental factors and the genotype-to-phenotype map.

Maternal CNV transmission to sons with autism correlates with phenotypic traits in the Broad Autism Phenotype

Autism Spectrum Disorder (ASD): Common neurodevelopmental disorder, global prevalence ~1 %; Persistent deficits in social communication and social interaction; restricted and repetitive behavior, interests, or activities – highly heterogeneous clinical presentation; Male to female ratio ~4:1.

TRANSFORMATIONS OF TASK-DEPENDENT PLASTICITY FROM A1 TO HIGHER-ORDER AUDITORY CORTEX

Everyday, humans and animals navigate complex acoustic environments, where multiple sound sources overlap. Somehow, they effortlessly perform an acoustic scene analysis and extract relevant signals from background noise. Constant updating of the behavioral relevance of ambient sounds requires the representation and integration of incoming acoustical information with internal representations such as behavioral goals, expectations and memories of previous sound-meaning associations. Rapid plasticity of auditory representations may contribute to our ability to attend and focus on relevant sounds. In order to better understand how auditory representations are transformed in the brain to incorporate behavioral contextual information, we explored task-dependent plasticity in neural responses recorded at four levels of the auditory cortical processing hierarchy of ferrets: the primary auditory cortex (A1), two higher-order auditory areas (dorsal PEG and ventral-anterior PEG) and dorso-lateral frontal cortex. In one study we explored the laminar profile of rapid-task related plasticity in A1 and found that plasticity occurred at all depths, but was greatest in supragranular layers. This result suggests that rapid task-related plasticity in A1 derives primarily from intracortical modulation of neural selectivity. In two other studies we explored task-dependent plasticity in two higher-order areas of the ferret auditory cortex that may correspond to belt (secondary) and parabelt (tertiary) auditory areas. We found that representations of behaviorally-relevant sounds are progressively enhanced during performance of auditory tasks. These selective enhancement effects became progressively larger as you ascend the auditory cortical hierarchy. We also observed neuronal responses to non-auditory, task-related information (reward timing, expectations) in the parabelt area that were very similar to responses previously described in frontal cortex. These results suggests that auditory representations in the brain are transformed from the more veridical spectrotemporal information encoded in earlier auditory stages to a more abstract representation encoding sound behavioral meaning in higher-order auditory areas and dorso-lateral frontal cortex.

Habitat selection and phenotypic diversity in the anchovy of the Bay of Biscay

Diversity among individuals in a population is an important feature linking vital rates with behaviour and spatial occupation. We measured the growth increments in the otolith of individual fishes collected on the annual fisheries survey PELGAS from 2001 to 2015. Individuals who grew larger at juvenile stage occupied later in life more off-shore habitats. Further, we analysed the allozymes of 13 different loci from 2001 to 2006. Alleles of the enzyme IDH showed different frequencies in inshore and offshore habitats. The population spatially segregates along a coast to off-shore gradient with individuals showing different early growth and allele frequencies. Results show how individuals in a population segregate spatially in different habitats in relation with phenotypic diversity. This implies modelling the population with individual-based and physiological approaches to fully grasp its dynamics. It also implies developing management strategies to conserve infra-population diversity as a means to garantee the occupation of the full range of habitats.

I. Quantal effects in biochemical cooperativity and a proposed mechanism for the differentiation of calcium signaling in synaptic plasticity. II. Evolutionary algorithms for the optimization of methods in computational chemistry

In Part 1 of this thesis, we propose that biochemical cooperativity is a fundamentally non-ideal process. We show quantal effects underlying biochemical cooperativity and highlight apparent ergodic breaking at small volumes. The apparent ergodic breaking manifests itself in a divergence of deterministic and stochastic models. We further predict that this divergence of deterministic and stochastic results is a failure of the deterministic methods rather than an issue of stochastic simulations.

Ergodic breaking at small volumes may allow these molecular complexes to function as switches to a greater degree than has previously been shown. We propose that this ergodic breaking is a phenomenon that the synapse might exploit to differentiate Ca$^{2+}$ signaling that would lead to either the strengthening or weakening of a synapse. Techniques such as lattice-based statistics and rule-based modeling are tools that allow us to directly confront this non-ideality. A natural next step to understanding the chemical physics that underlies these processes is to consider \textit{in silico} specifically atomistic simulation methods that might augment our modeling efforts.

In the second part of this thesis, we use evolutionary algorithms to optimize \textit{in silico} methods that might be used to describe biochemical processes at the subcellular and molecular levels. While we have applied evolutionary algorithms to several methods, this thesis will focus on the optimization of charge equilibration methods. Accurate charges are essential to understanding the electrostatic interactions that are involved in ligand binding, as frequently discussed in the first part of this thesis.

Influence of Acute and Chronic Exercise on Markers of Hippocampal Plasticity

Exercise and physical activity are lifestyle behaviors associated with enriched mental health. Understanding the mechanisms by which exercise and physical activity improve mental health may provide insight for novel therapeutic approaches for numerous mental health disorders. This dissertation reports the findings from three studies investigating the influence of acute and chronic exercise on behavioral and mechanistic markers of hippocampal plasticity and delves into the potential role of noradrenergic signaling in the hippocampal adaptations with exercise. The first study assessed the effects of long-term voluntary wheel running on hippocampal expression of plasticity-associated genes and proteins in adult male and female C57BL/6J mice, highlighting sex differences in the adaptations to long-term voluntary wheel running. The second study examined the influence of acute exercise intensity on AMPA receptor phosphorylation, a mechanism essential for hippocampal plasticity, plasticity- associated gene expression, spatial learning and memory, and anxiety-like behavior. The unexpected finding that acute exercise increased anxiety-like behavior encouraged investigation into the role of central noradrenergic signaling in acute exercise-induced anxiety. The third study determined how previous exposure to voluntary wheel running modulates the response to an acute bout of exercise, focusing primarily on transcription of the important plasticity-promoting gene, brain-derived neurotrophic factor. Using a pharmacological approach to compromise the locus coeruleus noradrenergic system, a system that is implicated in age-related mental health disorders such as Alzheimer’s Disease, the third study also investigated the influence and interaction of the noradrenergic system and acute exercise on expression of multiple brain-derived neurotrophic factor transcripts. Together, this dissertation reports the findings from a series of experiments that explored similarities, differences, and interactions between the effects of acute and chronic exercise on markers of hippocampal plasticity and behavior. Further, this work provides insight into the role of the noradrenergic system in exercise-induced hippocampal plasticity.

Pathogenicity and phenotypic sulfadiazine resistance of Toxoplasma gondii isolates obtained from livestock in northeastern Brazil

Toxoplasma gondii is the causative protozoan agent of toxoplasmosis, which is a common infection that is widely distributed worldwide. Studies revealed stronger clonal strains in North America and Europe and genetic diversity in South American strains. Our study aimed to differentiate the pathogenicity and sulfadiazine resistance of three T. gondii isolates obtained from livestock intended for human consumption. The cytopathic effects of the T. gondii isolates were evaluated. The pathogenicity was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using a CS3 marker and in a rodent model in vivo. Phenotypic sulfadiazine resistance was measured using a kinetic curve of drug activity in Swiss mice. IgM and IgG were measured by ELISA, and the dihydropteroate synthase (DHPS) gene sequence was analysed. The cytopathic effects and the PCR-RFLP profiles from chickens indicated a different infection source. The Ck3 isolate displayed more cytopathic effects in vitro than the Ck2 and ME49 strains. Additionally, the Ck2 isolate induced a differential humoral immune response compared to ME49. The Ck3 and Pg1 isolates, but not the Ck2 isolate, showed sulfadiazine resistance in the sensitivity assay. We did not find any DHPS gene polymorphisms in the mouse samples. These atypical pathogenicity and sulfadiazine resistance profiles were not previously reported and served as a warning to local health authorities.