This dissertation is focused on how circadian control of olfactory responses are regulated at the cellular and molecular level in Drosophila. Electrophysiological approaches consisting of Electroantennogram (EAG), single unit recordings, among other techniques, were used to investigate the extent of autonomy of peripheral oscillators from central pacemaker cells, the molecular targets of the circadian oscillator in antennal neurons, and the nature of circadian influence on single unit responses recorded from basiconic sensillae. To address the question of cellular mechanisms mediating olfaction rhythms, UAS-Gal4 strategies were used for tissue specific expression of dominant negative forms of CLK (CLOCK) and CYC (CYCLE). Specifically, OR (Odorant Receptor)- GAL4 constructs were used to achieve cell specific expression in the antenna. By recording EAG responses from specific regions of the antenna, it was found that antennal sensory neurons possess independent oscillators that are both necessary and sufficient to drive rhythms in olfactory responses. To understand the molecular mechanisms controlling olfaction rhythms, the effect of GRK-2 and an arrestin (KURTZ) of the olfactory signal transduction pathway were studied by use of respective mutants and the effect of cell specific rescue of these proteins by UAS-Gal4 approaches was also documented. Interestingly, these molecules have phenotypes that argue for a different role of for these proteins in Drosophila olfaction as opposed to their respective functions in vertebrate systems. Finally, single unit recordings were measured from different basiconic sensilla and the influence of the circadian oscillator was studied on select parameters of the single unit data obtained. It was found that spike amplitude of the spontaneous response was the only parameter under circadian control and that these rhythms are dependant on input from the odorant receptor activated pathway.

The avian circadian system is composed of multiple inputs, oscillators and outputs. Among its oscillators is a hypothalamic structure presumed to be homologous to the primary circadian pacemaker in mammals, the suprachiasmatic nucleus (SCN). The SCN in avian species is poorly defined: two structures in the hypothalamus, the medial SCN (mSCN) and visual SCN (vSCN), have been referred to in the literature as the SCN. The present studies were designed to answer one central question: where is the avian homolog to the mammalian SCN? Uptake of 2-[14C]-deoxyglucose (2DG), an indicator of glucose metabolism, fluctuates in the mSCN and vSCN in both a daily and circadian manner. These data indicate a possible role in the circadian system for both the vSCN and the mSCN. Additionally, several visual structures display daily fluctuations of 2DG uptake, two of which exhibit circadian variations, supporting previous studies indicting circadian regulation of the visual system. Efferents and afferents of the mSCN and vSCN were identified and compared to those of rodents. While the mSCN bears a stronger resemblance to the rodent SCN in its efferent connections than the vSCN, afferents of both are comparable. The total number of mSCN and vSCN neuronal connections far exceeds that of the rodent SCN. A subset of these connections is strikingly similar to those of the rodent SCN, while others are found to connect these two nuclei to the visual system. These data further support the involvement of both the mSCN and vSCN in the circadian and visual systems. Suprachiasmatic organization was addressed using classical techniques. Though loosely similar in location to the mammalian SCN, the mSCN is cyto- and chemoarchitecturally different, while the vSCN bears more similarity to the mammalian SCN in this regard. A unique astrocytic bridge exists between the mSCN and vSCN, suggesting a role for astrocytes in the circadian system. Finally, the vSCN efferent to the medial nucleus of Edinger-Westphal was verified using a technique that may advance future studies of avian of circadian organization. The current data and the available literature were considered in the development of a working model of the avian SCN.

Circadian clocks are composed of molecular oscillators and are found in most eukaryotes and some prokaryotes. The fungus Neurospora crassa is a leading model for studying the clock. In N. crassa, the well-described FRQ/WCC Oscillator (FWO) consists of a molecular feedback loop involving the negative element FRQ, the blue-light photoreceptor WC-1, and WC-2. WC-1 and WC-2 form a complex called WCC, which functions as the positive element in the feedback loop. The FWO is considered to be the core oscillator regulating overt rhythmicity. However, several labs have shown that rhythms can persist in the absence of a functional FWO under certain growth conditions and genetic backgrounds, suggesting the presence of additional oscillators in the cell. Using genetic approaches to identify components of these putative oscillators, we uncovered a mutant strain, called light-mutant 1 (LM1) that is robustly rhythmic in constant light, and in strains carrying deletions of FWO components; both of which are conditions that abolish FWO function. The oscillator (called the Light Mutant Oscillator, LMO) revealed in the LM1 mutant strain meets two of the three criteria for a circadian oscillator. The LMO has a free running period of ~ 24h, and it is temperature-compensated. However, while the LMO can respond to light cues, WC-1 is required for circadian entrainment to 24-h light cycles. The response of LM1 cells lacking the circadian blue-light photoreceptor WC-1 to blue-light suggested that alternate light inputs function in LM1 mutant cells. I show that the blue light photoreceptors VIVID and CRY compensate for each other, and for WC-1, in LMO light responses. Importantly, I show that deletion of the cryptochrome (cry) gene abolishes rhythmicity in an LM1 strain, providing evidence for a role for CRY in the clock mechanism. The LM1 mutation is recessive, suggesting loss of function. Therefore, we hypothesize that the LM1 gene encodes a protein that negatively regulates the activity of the LMO. Our mapping and sequencing data have placed the LM1 mutation on the left arm of chromosome I, near the mating type locus; however, the identity of the mutated gene remains elusive.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [84]-95).

No description available.

Thesis (Ph. D.)--University of California, San Diego, 2009. / Title from first page of PDF file (viewed July 23, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 127-136).

Circadian Rhythms are the outputs of an internal biological clock common to all animals. Most circadian rhythms have about a 24-hour period and can synchronize with the earth’s solar day. Our lab primarily studies the circadian rhythms of spiders because their internal clocks can be extremely different from 24 hours (some as much as five hours different), yet they can remain synchronized with the solar day. The objective of this study is to identify a non-spider species for comparison with spiders that, a) has a clear and measurable circadian rhythm, and b) can easily be bred and reared in the lab. Gromphadorhina Grandidieri, also known as Black Tiger Hissing Cockroach (hereafter referred to as ‘hissers’), is one of the very many species of African Hissing Cockroaches native to rainforests in Madagascar. These species get the nickname ‘hisser’ because of their defense mechanism that forces air through spiracles which are breathing holes in the exoskeleton to make a hissing noise like a cat would. These animals are colonial and are easily bred and reared in captivity. Our hypotheses were that G. Grandidieri will be nocturnally active with a typical animal circadian rhythm of close to 24 hours. To carry out this experiment we measured locomotor activity in Juvenile and adult hissers for 5 days in a 12:12 hour light:dark cycle followed by 7-10 days in constant darkness to reveal the animals’ internal clock period. We measured this using an activity monitor which records when an individual moves across an infrared light beam. Data were taken from 32 individuals in this manner. Our results showed that juveniles had a mean circadian rhythm of 24.03 + 0.49 hours, and that adults had a mean rhythm of 24.12 + 0.90 hours. Most, but not all, individuals had a clear and measurable circadian rhythm and survived the duration of the experiment. Locomotor activity in the light:dark cycle was mainly in the dark phase, confirming that these animals are generally nocturnal. Overall, our hypotheses were supported and we have identified an excellent comparison species for future research in our lab.

Circadian clocks are vital to the proper functioning of organisms’ internal processes and behavioral outputs and typically have endogenous periods that approximate (within 1-2 hours) the 24-hour solar day. Clocks that deviate significantly from about 24 hours are often associated with metabolic syndromes or other disease states. For instance, organisms with near-24-hour clocks have higher survivorship under 24-h light:dark (LD) cycles than with 22- or 26-hour cycles. Likewise, mutant organisms with 22-hour clocks survive better under 22-h cycles but fare poorly under 24- and 26-h cycles. In other words, organisms suffer if their circadian clocks do not “resonate” with environmental cycles. Organisms fail to synchronize (entrain) their activity with non-resonant LD cycles and this failure typically leads to a number of physiological disruptions. Interestingly, several spider species have endogenous circadian periods that deviate by several hours from the period of the Earth’s solar day. The object of the present study is to investigate whether the phenomenon of circadian resonance also pertains to these atypical spider circadian rhythms. We investigated three spider species, two of which have internal periods (τ) significantly different from 24 hours. Approximately 50 individuals of each species of spider (Frontinella communis: τ=29.05±0.62 hours; Metazygia wittfeldae: τ=22.74±0.24h; and Cyclosa turbinata: τ=18.54±0.28h) were placed into chambers with periods of 19 (9.5:9.5h L:D), 24 (12:12h L:D), or 29 hours (14.5:14.5h L:D). If resonance is pertinent for spiders, we would expect survivorship to decrease in non-resonant LD cycles. Instead, no spider species exhibited decreased longevity in non-resonant L:D cycles. These findings contradict all previous research into circadian resonance and suggest that spiders do not suffer the costs of extreme desynchronization. In a second experiment, 10-11 spiders from each species were placed into infrared activity monitors to determine if their locomotor activity could entrain to (synchronize with) the three different LD cycles. Individuals from all three spider species entrained to all LD period lengths, again in contrast with prior research in other species. These results indicate that spider circadian clocks have highly unusual limits of entrainment and suggest a remarkable level of plasticity in their release from the selective pressure to maintain an internal period of approximately 24 hours.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 153-163).

Daily rhythms of physiology and behaviour in mammals are orchestrated by a hierarchical network of cellular oscillators. The master pacemaker that defines local and systemic timing across the brain and body are the suprachiasmatic nuclei of the hypothalamus (SCN). Disruption to the timing of sleep and daily behavioural activity can manifest in a range of pathologies including neuropsychiatric disorders. Bipolar disorder (BPD) is once such neurological condition that exhibits profound associations with altered circadian rhythm generation and whose toolkit of pharmacological interventions impact upon circadian rhythm generation. Currently it is unclear exactly how changes to rhythmic physiology contribute to the aetiology and pathology of BPD. In recent years, rodent models possessing lesions within genes that make up the basic cellular oscillator are widely reported to exhibit concomitant changes in affective behaviours, namely mania-like phenotypes. Recently a mouse model possessing a mutation within the neuron-specific Na+/K+-ATPase (NKA) alpha3 subunit, known as Myshkin, was described as a model of the manic phase of BPD. The NKA alpha3 is not reported as a critical element of the circadian oscillator and we used this opportunity to characterise the behavioural and physiological circadian system of these animals. Under wheel-running paradigms Myk/+ animals exhibited a broad array of behavioural deficits including lengthened, low amplitude and labile free-running rhythms, altered phase re-setting and elevated metabolic activity. Physiological characterisation of the SCN revealed deficits in amplitude of electrical output and changes to post-synaptic signalling although the ex vivo molecular pacemaking of the SCN remained intact. Myshkin animals therefore represent a novel model in which changes to central output arise independently of changes to basic molecular pacemaking. Despite this seemingly distinct mechanism Myshkin animals share many mood and circadian phenotypes with other clock gene models of affective behaviours highlighting that changes to pacemaking output of the SCN may be a critical factor across animal models exhibiting circadian and mood deficits. In addition, the impact of the mood stabiliser lithium, commonly prescribed in BPD, on cellular pathways within the SCN was investigated. Lithium consistently lengthens the period of cellular and behavioural rhythms in mammals although the mechanism of this action is yet undefined. Glycogen synthase kinase 3β (GSK3β) and inositol monophosphatase (IMPase) are the major biochemical targets of lithium at therapeutic concentrations. GSK3β is known to shorten rhythms and this study targeted IMPase and inositol phosphate turnover in the period lengthening effects of lithium. We reveal that although inhibition of IMPase dampens SCN molecular rhythms, the period of oscillations remains unchanged and therefore lithium acts upon distinct cellular pathways within the SCN to exert effects on period.