Detection, counting, and classification of visual ganglia columns of drosophila pupae

Many neurobiologists use the fruit fly (Drosophila) as a model to study neuron interaction and neuron organization and then extrapolate this knowledge to the nature of human neurological disorders. Recently, the fluorescence microscopy images of fruit-fly neurons are commonly used, because of the high contrast. However, the detection of the neurons or cells is compromised by background signals, generating fuzzy boundaries. As a result, it is still common that in many laboratories, the detection, counting, and analysis of this microscope imagery is still a manual task. An automated detection, counting, and morphological analysis of these images can provide faster data processing and easier access to new information. The main objective of this work is to present a semi-automatic detection-counting system and give the main characteristics of images of the visual ganglia columns in Drosophila. We present the semi-automatic detection, count, segmentation and we concluded that it is possible to obtain an accuracy of 75% (with a Kappa statistic of 0.50) in the shape classification. Additionally, we develop python GUI CC Analyzer which can be used by neurobiology laboratories whose research interests are focused on this topic.Peer ReviewedPostprint (published version

Characterization of the Blackpatch mutation in Drosophila melanogaster

Mutations at the Blackpatch locus cause death and degeneration in the developing visual system of Drosophila melanogaster. Some Bpt alleles cause recessive lethality in the larval stage during development. To understand the role of Bpt in visual system development, the mutant phenotype was characterized for its effects on structure of the developing eye and the electrical activity of adult mutant eyes. The first hypothesis was that Bpt gene function may be required for the visual transduction pathway to receive and process light signals correctly. This is an electrical/biochemical process that can be visualised as an electroretinogram (ERG) and mutations in the pathway lead to abnormal ERGs. Bpt mutations may affect this pathway causing abnormal electroretinograms (ERGs). Analyses of the electroretinograms of mutant flies showed that mutant flies receive and process light correctly. Bpt does not function in the visual transduction pathway. The second hypothesis was that Bpt functions in a neurotrophic pathway. Genes in this pathway interact with signal transduction pathways necessary for cell-cell signaling and exhibit temporal and spatial specificity. Mutations in this pathway characteristically cause cell death in an apoptotic manner. In Bpt mutants, cell death in the developing eye occurs in a spatial and temporally specific manner suggesting a similar specificity for the action of the Bpt gene. The genetic focus of the mutation is in the lamina which is seen first around 42 hours post pupariation (pp) followed by death in the retina around 58 hours pp. Previous analyses has shown that Bpt interacts with Notch , an important cell signaling molecule that communicates by sending lateral inhibitory signals to its neighbors. The Bpt mutation affects the eye at a time of axonal innervation of the lamina. In mammalian systems, this phase involves the action of neurotrophic genes. Several lines of evidence suggest that Bpt may indeed be working in a neurotrophic pathway to maintain the laminar cells as they complete development;In the developing fly, Bpt mutations cause lethality in the homozygous condition. The lethal phase for Bpt is between 36 to 48 hours during the larval II instar phase of development. This suggests a time when the Bpt gene product is needed. The lethal phase is at a time of proliferation and development in the nervous system. Our results show that the morphology of the nervous system is normal at the end of embryogenesis. Bpt is necessary at a very specific time in development and any abnormalities due to Bpt take place during this time window. It is possible that the systemic death of the larva is due to multiple primary and secondary effects of the Bpt mutation. We suggest that Bpt has a vital function and this may be involved in the proliferation of the central brain neuroblasts during development;Blackpatch plays an important role in the development of Drosophila. In the adult visual system development it may be acting within a neurotrophic pathway that is necessary for maturation and maintenance when the retinal axons are innervating the lamina. In the larva, Bpt may again be functioning as part of a neurotrophic pathway in central nervous system development

Novel morphological and physiological scaling relationships in the southern red wood ant

Red wood ants (Formica rufa) are visual navigators whose colonies contain workers that differ substantially in size. By investigating the allometry of the ants’ compound eyes, and the regions within them, I showed that facets in particular regions scaled differently: both grade and slope shifts occurred. Facets in some eye regions were absolutely larger than others, while other facet regions scaled at different rates with body size. I next compared eye scaling between nests from the same population. Nevertheless, the method by which ants increased their eye size differed between nests. I found that ants from some nests primarily increased eye size through facet number and others through facet diameter. This showed that scaling rules at the cellular levels can differ even within a single population. Comparisons among Formica species revealed that differential eye scaling was not restricted to just F. rufa. Differential scaling was found in F. sanguinea but not F. lugubris or F. fusca. Surprisingly, scaling between facet diameter and number was conserved across all four species, demonstrating that whole-organ scaling among species can be conservative whilst differing vastly between organ-regions. Moving beyond morphology, I next investigated whether physiological scaling was equally as variable among nests. Metabolic rate scaling was negatively allometric and the same among four nests. Respiratory water loss was found to be determined solely by metabolic rate. Metabolic rate co-varies with different ventilation types, however, switches in ventilation type are driven by movement. This demonstrates that increases in metabolic rate are not sufficient to explain changes in ventilation type but are sufficient to explain respiratory water loss

Dopaminergic and Activity-Dependent Modulation of Mechanosensory Responses in Drosophila Melanogaster Larvae

A central theme of this dissertation is nervous system plasticity. Activity-dependent plasticity and dopaminergic modulation are two processes by which neural circuits adapt their function to developmental and environmental changes. These processes are involved in basic cognitive functions and can contribute to neurological disorder. An important goal in modern neurobiology is understanding how genotypic variation influences plasticity, and leveraging the quantitative genetics resources in model organisms is a valuable component of this endeavor. To this end I investigated activity-dependent plasticity and dopaminergic modulation in Drosophila melanogaster larvae using neurobiological and genetic approaches. Larval mechanosensory behavior is described in Chapter 2. The behavioral experiments in that chapter provide a system to study mechanisms of plasticity and decision-making, while the electrophysiological characterization shows that sensory-motor output depends on neural activity levels of the circuit. This system is used to investigate activity-dependent plasticity in Chapter 3, i.e., habituation to repetitive tactile stimuli. In Chapter 4, those assays are combined with pharmacological manipulations, genetic manipulations, and other experimental paradigms to investigate dopaminergic modulation. Bioinformatics analyses were used in Chapter 5 to characterize natural genetic variation and the influence of single nucleotide polymorphisms on dopamine-related gene expression. The impact and suggested future directions based on this work are discussed in Chapter 6. Dopamine also modulates cardiomyocytes. Chapter 7 describes biochemical pathways that mediate dopaminergic modulation of heart rate. The final two chapters describe neurobiology research endeavors that are separate from my work on dopamine. Experiments that have helped characterize a role for Serf, a gene that codes for a small protein with previously unknown function, are described in Chapter 8. In the final chapter I describe optogenetic behavioral and electrophysiology preparations that are being integrated into high school classrooms and undergraduate physiology laboratories. Assessment of student motivation and learning outcomes in response to those experiments is also discussed

A P Element Excision-Derived Mutation in Drosophila melanogaster

The purpose of the work described in this Thesis was to isolate and mutate novel genes essential in the development of the Drosophila melanogaster nervous system. Two marked P element enhancer trap strains were found to express the reporter gene lacZ in spatially and temporally regulated patterns in the nervous system. Excisions of the P element construct in one of these strains, A22, were detected in approximately 68% of the A22-derived progeny which were recognised by the loss of the rosy gene. 19 strains were subsequently found to have a recessive mutant phenotype believed to be a result of imprecise excision of the P element. This represents a mutation rate of approximately 24%. Each of these 19 strains had a recessive mutation that resulted in abnormal wing development. Other mutant phenotypes were not detected in any of the excision-derived strains. No similar mutations are known to be located at the site of P element insertion of the ancestral A22 strain and therefore these excision-derived mutants are considered novel. Four excision-derived mutants were selected for further characterisation and these were found to be cold-sensitive. The severity and frequency of the mutant phenotype was increased when these four strains were raised at 2