The fruit fly Drosophila exhibits robust daily behavioral rhythms, which are driven by a network of circadian pacemaker neurons in the fly brain. The Narrow Abdomen (NA) sodium leak channel functions rhythmically in pacemaker neurons, downstream of the molecular circadian clock, to depolarize resting membrane potential and promote neuronal excitability. Loss of NA function (NA-LOF) strongly disrupts behavioral rhythms, and these behavioral phenotypes are consistent with decreased circadian neuronal activity. Yet despite some recent advances, the mechanisms of NA channel function and regulation in the circadian system are still not well understood.
To further elucidate the role of the NA channel in the circadian neuronal network, we generated mutated versions of the NA transgene and assessed the effects of transgene expression in Drosophila circadian pacemaker neurons. Expression of a putative gain of function na transgene (na-GOF) in pacemaker neurons generates unique behavioral phenotypes, suggesting novel effects on neuronal excitability or/and the molecular circadian clock. Next, we investigated how NA-LOF and NA-GOF mutations affect circadian neuron activity through optical recording of fluorescent voltage and calcium sensors expressed in these neurons. Using the fluorescent voltage sensor ArcLight, we find that both NA-LOF and NA-GOF manipulations suppress spontaneous membrane activity in clock neurons in the Drosophila brain. This finding was surprising because the behavioral effects of NA-LOF and NA-GOF are quite distinct. However, the information provided from these spontaneous assays may be a combination of neuronal input and output, and in some cases information is combined from multiple cells. To further characterize the neurophysiological effects of NA channel manipulation, we next paired optical recording with pharmacology in brain explants. Here we find that both wild-type and NA-LOF DN1p clock neurons are strongly depolarized by the acetylcholine receptor agonist nicotine, while NA-GOF neurons show little response. This suggests that NA-GOF expression already depolarizes the membrane potential of these neurons. We also assessed intracellular calcium levels in the DN1p clock cells after applying the inhibitory neurotransmitter glutamate at either morning (peak) or evening (trough) timepoints. We find that wild-type DN1p neurons show a strong decrease in calcium at the peak timepoint and a much smaller decrease at the trough. In contrast, NA-GOF DN1p neurons show decreases at both timepoints, indicating that they have elevated calcium levels (and elevated activity) at the trough time. Through immunostaining, we find that NA-GOF expression alters the core clock protein PERIOD levels in sLNv and LNd neurons during early day. Taken together, this study shows that overexpression of NA-GOF ion channel in Drosophila pacemaker neurons induce unique behavioral phenotypes, likely by depolarizing membrane potential and increasing neuronal activity. We propose that these changes in neuronal activity may feedback to alter the oscillation of molecular clocks.
While these transgenic studies have been informative, we have also established gene-editing methods in order to distinguish the effects of gene mutation from effects of overexpression. We have used the CRISPR-Cas9 system to target the endogenous na locus. In the initial step, we replaced na exons 1-13 with a fluorescent marker flanked by attP integration sites. Through subsequent integrase-mediated recombination, we hope to generate a series of na mutations of interest, including gain-of-function mutations, for future studies.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-7963 |
| Date | 01 August 2018 |
| Creators | Lu, Xinguo |
| Contributors | Lear, Bridget |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright © 2018 Xinguo Lu |
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