Macleod, Gregory

Learning and memory studies in Drosophila melanogaster have led to advances in understanding fly and mammalian genetics and neurophysiology. Despite extensive studies, there remain gaps in the scientific literature concerning genes and neural pathways involved in learning and memory. There are differences in the memory traces between olfactory and visual memory, yet visual learning has not been studied to the same extent as olfactory learning. Visual place learning has only recently been addressed. We offer a new apparatus for studying visual place learning in D. melanogaster. The new apparatus offers a compelling and cost-effective approach to investigating visual place learning. The most notable difference between the new apparatus and others designed for visual place learning is the use of Peltier units in conjunction with a hydraulic system to supply heat used as a negative stimulus, with the advantages of even heat distribution, ease of construction, and ease of operation.

Parkinson’s Disease (PD) is a neurodegenerative disorder that affects millions of
people around the world, although it is more common in individuals aged 60 years or
older. PD is associated with the degeneration of dopamine neurons in the substantia
nigra. While the underlying cause of neuronal degeneration is poorly understood,
mitochondrial dysfunction is a common feature of the cell pathology. Geneticallyencoded
fluorescent probes were used to label the mitochondria in wildtype Drosophila
melanogaster and those genetically manipulated to model PD. Brains were dissected,
immunolabeled, and their mitochondria were imaged using structured illumination
microscopy (SIM). Mitochondrial number was determined, as well as the sphericity and
surface area quantified. This characterization of mitochondrial number and morphology
in wildtype Drosophila created a baseline for comparison to Drosophila that over-express
the wildtype human α-synuclein protein which is associated with PD.

Autism Spectrum Disorder (ASD) can be can be caused by numerous different genetic abnormalities, one of which is Pten haploinsufficiency. There is some evidence to suggest that environmental enrichment can decrease the symptoms of ASD and Pten mutant mice have been shown to have altered social behaviors. Therefore, Pten and WT mice were raised either in standard or environmentally enriched cages and these mice were then tested for social recognition. WT females in both environmentally enriched and standard cages and Pten males raised in environmentally enriched cages can recognize and distinguish between other mice. Pten females raised in both standard and environmentally enriched cages, and Pten males in standard cages did not show statistically significant recognition. WT males in both environmentally enriched and standard cages also lacked significant recognition. This outcome indicates that either the experimental protocol should be re-examined or that more mice are required for the experiment.

Dermatophilus congolensis is a gram positive, non-acid fast, facultative anaerobic actinomycetes that causes an epidermal skin infection in bovine, ovine, and equine species. This thesis studies the inhibitory effects of common antibacterial topical products on Dermatophilus congolensis. An initial experiment was performed on equine subjects and a secondary experiment was performed using a live strain of the bacteria. Seven different topical products were used in the final experiment, each showing some level of growth inhibition. Chlorhexidine 2% scrub was by far the most potent product with the greatest growth inhibition in each experiment. The use of a topical product such as Chlorhexidine is not only effective, but it helps to reduce bacterial resistance.

Phosphatase and tensin homologue (PTEN) is a gene that, when mutated, can
cause macrocephaly/autism syndrome. The Pten mutant mouse model will help identify
genetic modifiers of Pten-related neurodevelopmental phenotypes, with the goal of
gaining insight into the polygenic nature of autism. We hypothesize that genes that
display spatiotemporal coexpression patterns similar to Pten in the developing brain are
candidates to genetically interact with Pten. Fbxw7, has been identified as a strong
candidate. We have conditionally deleted Fbxw7 (Fbxw7 cKO), Pten (Pten cHet), and
Pten and Fbxw7 together (Pten and Fbxw7 double mutant) in the developing cerebral
cortex. We found Fbxw7 cKO mice have decreased cortical mass and cell number,
increased cell density, hydrocephalus and premature lethality. Pten cHet mice display
increased cortical mass and cell number, with unchanged cell density and no
hydrocephalus or premature lethality. Strikingly, Pten and Fbxw7 double mutant mice
had the exact phenocopy of Pten cHet mice, indicating a surprising epistatic interaction
between Pten and Fbxw7, in which Pten overrides the effects of Fbxw7. Further work
will explore the mechanism of this interaction and will characterize cortical phenotypes
in mutant animals.

Mitochondrial dysfunction has been associated with the pathology of most neurodegenerative diseases. An essential element of mitochondrial function is a robust proton motive force (PMF) across the inner mitochondria membrane (IMM).

The purpose of this project was to understand the effects of therapeutic antidepressants with respect to circadian rhythm in Drosophila melanogaster. Antidepressants are known to have a role in dopamine and serotonin signaling pathways. These pathways have been observed to have a role in circadian rhythm, the biological process involving sleep patterns. In the experiments completed thus far, it has been observed that the flies administered antidepressants have more fractioned sleep than the control group flies. It has also been noted that normal light to dark sleep cycles are altered significantly in the flies given antidepressants. It is important to research and to understand the effects of antidepressants in Drosophila melanogaster because it could lead to a more effective way to administer antidepressants to humans without harmful side effects.

Changes in synaptic strength underlie synaptic plasticity, the cellular substrate for learning and memory. Disruptions in the mechanisms that regulate synaptic strength closely link to many developmental, neurodegenerative and neurological disorders. Release site probability (PAZ) and active zone number (N) are two important presynaptic determinants of synaptic strength; yet, little is known about the processes that establish the balance between N and PAZ at any synapse. Furthermore, it is not known how PAZ and N are rebalanced during synaptic homeostasis to accomplish circuit stability. To address this knowledge gap, we adapted a neurophysiological experimental system consisting of two functionally differentiated glutamatergic motor neurons (MNs) innervating the same target. Average PAZ varied between nerve terminals, motivating us to explore benefits for high and low PAZ, respectively. We speculated that high PAZ confers high-energy efficiency. To test the hypothesis, electrophysiological and ultrastructural measurements were made. The terminal with the highest PAZ released more neurotransmitter but it did so with the least total energetic cost. An analytical model was built to further explore functional and structural aspects in optimizing energy efficiency. The model supported that energy efficiency optimization requires high PAZ. However, terminals with low PAZ were better able to sustain neurotransmitter release. We suggest that tension between energy efficiency and stamina sets PAZ and thus determines synaptic strength. To test the hypothesis that nerve terminals regulate PAZ rather than N to maintain synaptic strength, we induced sustained synaptic homeostasis at the nerve terminals. Ca2+ imaging revealed that terminals of the MN innervating only one muscle fiber utilized greater Ca2+ influx to achieve compensatory neurotransmitter release. In contrast, morphological measurements revealed that terminals of the MN inner vating multiple postsynaptic targets utilized an increase in N to achieve compensatory neurotransmitter release, but this only occurred at the terminal of the affected postsynaptic target. In conclusion, this dissertation provides several novel insights into a prominent question in neuroscience: how is synaptic strength established and maintained. The work indicates that tension exists between energy efficiency and stamina in neurotransmitter release likely influences PAZ. Furthermore, PAZ and N are rebalanced differently between terminals during synaptic homeostasis.