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dc.contributor.advisorSchulz, David J.eng
dc.contributor.authorBerigan, Bentoneng
dc.date.embargountil5/31/2023
dc.date.issued2022eng
dc.date.submitted2022 Springeng
dc.description.abstractAnimal research models provide the opportunity to decode neural activity via the ability to precisely stimulate and record neural events associated with quantifiable circuit and behavioral outputs (Bernstein et al., 2012; Boyden et al., 2005; Deisseroth et al., 2006; Roth, 2016). As neuroscientists continue to decode the nervous system, a collection of experimental tools to stimulate and record neural activity in real-time that are amenable to a variety of scenarios and model organisms both in vitro and in vivo is invaluable for basic research and translational work. Here, we sought to develop tools to simultaneously measure and manipulate neural activity in intact nervous systems with relevant behavior activity, ultimately broadening the use of these technologies. Additionally, we've laid the groundwork for deploying these tools using other experimental systems that provide additional basic and translational relevance. We investigated the thermosensitive properties of Drosophila Gr28bD, a recently discovered thermosensitive protein. We show that Gr28bD can be expressed in a heterologous system, and it generates a temperature dependent cationic current that it is not voltage sensitive. When expressed in Drosophila motor neurons, we observe temperature dependent increases in the frequency and intensity of calcium fluorescence signals measured using a genetically encoded calcium indicator, GCaMP6f. We also show that activation of Gr28bD is sufficient to elicit behavioral differences in flies with pan-neuronal and pan-motor neuron expression of Gr28bD. In summary, we validate Gr28bD as a novel thermogenetic tool as it can generate temperature dependent cationic currents that depolarize neurons, leading to behavioral changes. To further develop thermogenetic tools, we hypothesized that Drosophila species which inhabit diverse environments may contain naturally occurring thermosensitive ion channels with high sequence homology to Gr28bD yet unique temperature sensing properties. We tested Gr28bD orthologs from five Drosophila species: D. simulans (DsimGr), D. yakuba (DyakGr), D. psuedoobscura (DpseGr), D. willistoni (DwilGr), and D. mojavensis (DmojGr) using thermogenetics to exogenously express and activate Gr28bD orthologs in Drosophila motor neurons while recording neural activity via GCaMP6f. We found that DsimGr, DyakGr, DpseGr, and DwilGr are effective thermogenetic tools. However, these orthologs do not respond to temperature in the same manner. Flies overexpressing DwilGr show a similar temperature response to flies with overexpression of Gr28bD flies (37.5 - 38 degreesC), whereas DsimGr, DyakGr, and DpseGr flies are activated at lower temperatures (33 - 35 degreesC). Additionally, we show that DyakGr and DpseGr flies have a greater effect on neuronal activity compared to Gr28bD. Interestingly, DmojGr is not temperature sensitive below 40 degreesC, suggesting a difference in structure/function relative to other Gr28bD orthologs. This work further validates Drosophila Grs as a family of novel thermosensors, and it adds four new tools to the suite of available thermogenetic tools. To implement technologies for stimulating and recording neural activity in novel systems that have not been well characterized yet have significant basic and translational relevance, we focused on autonomic activity in the mouse bladder circuitry during the filling and voiding of the bladder. Despite bladder function in mice being functionally different than humans (Fowler et al., 2008), mouse bladder circuitry provides an ideal system to study the basic physiology of autonomic activity and its role in bladder function as parasympathetic and sympathetic signals are functionally and anatomically separate in this system (Wanigasekara et al., 2003). Moreover, we can directly evaluate bladder circuit output using cystometry to record bladder pressure (Ito et al., 2017). Rodent models of spinal cord injury (SCI) have shown that SCI recovery is dependent in part on plasticity of the autonomic pathways as well as bladder reflexes initiated by autonomic neurons (De Groat et al., 1998; de Groat and Yoshimura, 2006). We show that simultaneously recording parasympathetic activity via the pelvic nerve and sympathetic activity via the hypogastric nerve is technically challenging in mice. However, we were able to show for the first time that it is feasible to record from multiple peripheral nerves in the lower urinary tract during filling and voiding of the bladder. Therefore, we are optimistic that pelvic and hypogastric nerve recordings will be achievable with further optimizations. To record activity from peripheral neurons involved in bladder function, we establish in vivo fluorescent calcium imaging in the lower urinary tract during the filling and voiding of the mouse bladder using our custom miniscope and AAV serotypes that transduce peripheral tissue with higher efficiency. We are now poised to employ calcium imaging tools in major pelvic ganglion (MPG) neurons to study their normal activity during filling and emptying of the mouse bladder. Additionally, we demonstrate that Oregon Green BAPTA-1, AM (OGB-1, AM) can be used to measure calcium signals in response to pelvic nerve stimulation using whole-mount MPG preparations in vitro. However, the effective loading of OGB-1, AM into MPG neurons was a challenge in vivo because of the protective tissue surrounding MPG neurons and non-specific labeling of surrounding tissue. These approaches provide lots of information on normal autonomic activity during the filling and voiding of the mouse bladder at multiple levels: neurotransmission at autonomic ganglia, afferent and efferent autonomic activity in the lower urinary tract, and bladder circuit output. We are optimistic that we can use the tools in a translational context to understand how injury and disease impact autonomic signals in the lower urinary tract.eng
dc.description.bibrefIncludes bibliographical references.eng
dc.format.extentxii, 127 pages : illustrations (color)eng
dc.identifier.urihttps://doi.org/10.32469/10355/91654eng
dc.identifier.urihttps://hdl.handle.net/10355/91654
dc.languageEnglisheng
dc.publisherUniversity of Missouri--Columbiaeng
dc.relation.ispartofcommunityUniversity of Missouri--Columbia. Graduate School. Theses and Dissertationseng
dc.titleNeuroscience tools in novel contexts: developing thermogenetics in Drosophila and decoding autonomic activity in mouse major pelvic ganglioneng
dc.typeThesiseng
thesis.degree.disciplineBiological sciences (MU)eng
thesis.degree.grantorUniversity of Missouri--Columbiaeng
thesis.degree.levelDoctoraleng
thesis.degree.namePh. D.eng


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