The present work deals with several approaches to the study of regions near the centromere of chromosome 3 of Drosophila melanogaster. The goals of this research were: (i) to examine spontaneous crossing over near the centromere in detail; (ii) to locate the Deformed locus genetically and to determine whether this lesion is a recessive lethal; (iii) to test the efficacy of radiation-induced crossing over as a method of producing proximal aberrations; and (iv) to genetically and developmentally characterize a temperature-sensitive (ts) allele of a Minute locus, located near the centromere. CHAPTER 2 describes a study of recombination, which deals with short genetic regions near the centromere of chromosome 3, using the intervals, st-in-ri-eg²-Ki-p[sup P] and Gl-p[sup P]-Sb-H. The following generalizations have emerged: (i) an excess of multiple crossover chromosomes was recovered, and the intervals which immediately span the centromere showed the highest negative interference; (ii) a positive correlation of simultaneous exchange within closely-linked intervals, was noted for many of the multiple crossovers; and (iii) several classes of reciprocal crossover products were not recovered equally. Three possible explanations for these results are: pre-meiotic exchange, chromatid interference and gene conversion. The results of one experiment also indicated that the interchromosomal effects of C(1)M3 are most pronounced within the st-in and Ki-p[sup P] intervals. CHAPTER 3 describes a genetic study of the Deformed locus. The mapping results confirmed that Dfd is closely linked to Ki. Genetic analysis of the crossover chromosomes suggested that Dfd is homozygous viable and this was confirmed by the synthesis of homozygous Dfd stocks. This indicates that the Dfd locus is not located within section 84F in proximal 3R. CHAPTER 4 deals with experiments involving the use of radiation to produce crossovers near the centromere of chromosome 3, in males. Crossovers originating from exchange nearest the centromere, were associated with clusters more frequently than those originating from exchange within other proximally-adjacent segments. Induced exchange was frequently accompanied by mutation and/or chromosome damage, at or near the site of exchange. This was particularly true for crossovers resulting from exchange in wholly euchromatic segments. It is suggested that many of the radiation-induced crossovers arise through asymmetrical exchange, and that this approach will permit the isolation of proximal aberrations. CHAPTER 5 describes the genetic and developmental analysis of a ts Minute. As a ts allele of a proximally-located Minute locus, Q-III exhibits the classical dominant M traits, recessive lethality, and a highly pleiotropic phenotype, at 29°C. This phenotype was analysed in detail through the use of various temperature shift experiments. Q-III possesses a polyphasic temperature-sensitive period (TSP) for lethality extending from the first larval instar to late pupation. Shorter heat pulses defined discrete larval, larval/pupal, and pupal TSPs for lethality. In addition, homozygous Q-III females exhibit ts sterility and maternal effects, indicating that the Q-III gene product is essential throughout development. Heat-pulse experiments revealed a number of adult developmental abnormalities, involving derivatives of eye-antennal, leg, wing and genital imaginal discs. Many defects, for example, those involving the eye or antenna (eye-antennal disc), male genitalia (genital disc), and scutellum (wing disc), have larval TSPs; whereas others, such as bristle or sex comb traits, have pupal TSPs. It is suggested that the former defects may be related to cell death in the larval anlagen; while the latter are more likely due to blockages in differentiation during pupation. Q-III also interacts in ts fashion with several non-allelic mutations . Thus, at 29°C, Q-III is lethal when combined with DI, Ly and Dfd; suppresses the sex comb phenes of Msc and Pc; and produces wing nicking effects when combined with vg or Sex. TSPs were defined for the vg, Dl and Sex interactions. It is suggested that many of these interactions arec:metabolic rather than specific. The fact that Q-III phenotypically resembles bobbed and ts suppressor of forked[sup ts], strengthens the notion that Minute gene products are active in translation. It is concluded that translational defects can fully account for the pleiotropy of Q-III. / Science, Faculty of / Zoology, Department of / Graduate

Neuropeptides are central modulators of many functions including male-specific mating behaviours. Understanding how these chemical messengers modulate the neural substrates are still not well understood but remains important for biological research. In Drosophila melanogaster, two well-defined microcircuits (Longer-Mating-Duration (LMD) and Shorter-Mating-Duration (SMD)), are used to understand the underlying mechanisms of how neuropeptide interactions modulate temporal information in mating behaviours. In our study, we investigated the influence of SIFamide receptor-mediated signaling and its association to both LMD and SMD. We performed several RNAi-based screens where we identified and mapped out seven different types of neuropeptidergic neurons which were found to be important to either LMD and/or SMD. Following this analysis, we highlight three independent signaling pathways which are necessary to describe the cellular mechanics of the neuropeptides involved. Firstly, we infer that synaptic contacts between proctolin and SIFamide neurons in the subesophageal ganglion mediate inhibition in SMD whereas proctoclin as a neuropeptide modulates both LMD and SMD in a non-synaptic manner. Secondly, we describe an existing insulin-related microcircuit that is modulated by the inputs of Dimmed (DIMM), a transcription factor, through adipokinetic hormone, allatostatin A, and leucokinin to exhibit SMD. Thirdly and lastly, we discuss our interpretations of how capability neurons in the central brain resolves a potential disinhibition microcircuit in LMD via olfactory based signaling in the antennae lobe. In summary, our results contribute to establishing a model system to study neuropeptidergic microcircuits in complex mating behaviours.

Internal states, such as arousal and hunger, elevate the probability of a set of behaviors and persist on longer timescales than the behaviors that they predict. These states are triggered by sensors (e.g. neurotransmitters, biogenic amines) within the animal that detect internal homeostatic conditions and external factors. However, the sustained nature of internal states and the diversity of behaviors associated with a singular state suggest that state is represented not only by hormonal and modulatory signals but also by the coordinated activity of neurons within the central brain. Additionally, recent evidence suggests that internal states are represented throughout cortex in rodents and in many neuropil regions in Drosophila. In this thesis, I suggest how persistent states are represented globally in the brain by observing the activity of neurons, at the single-neuron level, distributed throughout the brain of Drosophila melanogaster and determining on what timescales their neural activity predicts behavior. To do this, we first establish a strategy to rapidly capture brain-wide activity of an awake, freely behaving Drosophila adult. We employ Swept Confocally Aligned Planar Excitation (SCAPE) microscopy, which has been shown to be an effective tool for volumetric imaging in a wide range of living samples, including zebrafish and Drosophila larvae. SCAPE's volumetric imaging speeds exceed those of point-scanning methods ten- to hundred-fold, and offers additional advantages, such as reduced phototoxicity and high signal-to-noise. The optical geometry of SCAPE consists of a single objective located directly above the sample. Therefore, this single stationary objective lens allows for imaging of intact, behaving animals like adult flies. Here, we characterize the spatial resolution of the system with respect to in vivo imaging of neurons in the adult fly brain. We show that we can achieve single-cell resolution, even in closely-spaced or dense neuronal populations. Additionally, we show that high-speed imaging of calcium activity throughout the whole brain can be performed at 20 fly brain volumes per second. These rates allow us to monitor neural dynamics occurring on the time scale of hundreds of milliseconds, which lets us capture the dynamics of popular calcium indicators like GCaMP. Moreover, we have demonstrated the feasibility of this approach to optically record odor responses of individual neurons in the olfactory circuit, while the animal freely behaves on a spherical treadmill. Having established a system for whole-brain imaging in Drosophila, we then use this methodology to explore the representation of two internal states: arousal, in flies freely running on a spherical treadmill, and hunger, in food-deprived flies consuming sugar. We define internal state as neural activity that predicts behavior on long timescales. To determine the timescale with which individual neurons best predict behavior, we define a regression model in which the activity of each cell is proportional to behavior filtered with unique time constant (tau_i). In freely running flies, we see that the neural activity exhibits a strikingly large dominant mode - nearly all cells across the brain are correlated with locomotion. While the median timescale is short, the distribution of timescales across all cells is broad, with some neurons correlated with locomotion on a much longer timescale, representing arousal based on our definition. In food-deprived flies fed sugar, no dominant mode exists; the neural activity tracking feeding is relatively subtle at the global scale. However, by applying the regression model to determine the timescales of individual cells, we do identify some ensembles of neurons possessing either a short timescale (tau_i < 10s), likely representing reward, or a long timescale (tau_i > 60s), putatively representing hunger. To investigate the populations that make up these different timescales, we used both genetic labeling and hierarchical clustering to determine the identity of neurons of interest. For example, in the freely running flies, we notice that cells in a dorsomedial region called the pars intercerebralis exhibit consistently large tau_i with respect to locomotion. Similarly, by genetically labeling neurons producing the hormone DH44, we see that in food-deprived flies consuming sugar, these neurons exhibit large tau_i with respect to feeding. Thus, we have identified dimensions of global dynamics, including a broadly distributed behavioral state as well as subspaces supporting putative neural correlates of the internal states of arousal and hunger. These data presented in this thesis, and the techniques we have established, have the potential to significantly impact our understanding of internal states at a global level in Drosophila melanogaster and can be extended to other organisms.

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