Ca²⁺ influx is one of the critical events that trigger synaptic vesicular release, and the accumulation of residual free Ca²⁺ in synapses is also important for activity-dependent synaptic plasticity. Ca²⁺ imaging with fluorescence indicators (synthetic or genetically encoded) is a powerful approach to monitor Ca²⁺ levels in neurons and synapses. Although accumulating studies in vertebrate systems have been carried out to demonstrate the role of Ca²⁺ in synaptic transmission and plasticity, most of these studies rely on pharmacological methods to infer the molecular mechanism, with less emphasis on forward genetic analysis. The Drosophila neuromuscular junction (NMJ) is a powerful neurogenetic platform for studying synaptic transmission, because of the availability of many mutations. However, not many mutations have been analyzed with Ca²⁺ imaging. Besides, although Genetically Encoded Ca²⁺ Indicators (GECIs) including GCaMPs are increasingly popular as the tool to identify neuronal circuits activated by certain stimuli or mediating particular behaviors, the physiological and functional interpretation of neuronal Ca²⁺ transients reported by GECIs remain obscure. By expressing GCaMPs in NMJ synapses, I characterized a spectrum of genetic mutations including sodium channel alleles parats¹, parabss¹, potassium channel mutations Shaker (ShM, Sh¹²⁰), Shab³, ether-a-go-go (eag¹, eag⁴pm), and double mutant eag¹ Sh¹²⁰. Drosophila NMJs contain at least three different types of synapses, which include glutamatergic tonic motor synapse type Ib, phasic motor synapse type Is, and modulatory octopaminergic synapse type II. In this study, I found that the ion channel mutations did not uniformly alter the Ca²⁺ dynamics in type Ib, Is and II synapses. Based on genetic dissection and pharmacological analyses, I concluded that the excitability type I and type II synapses are differentially regulated by various ion channels, and that ion channels mainly influence the influx of Ca²⁺ upon membrane depolarization but not the subsequent clearance. I also attempted to interpret the significance of synaptic Ca²⁺ transients by correlating Ca²⁺ imaging with electrophysiological recordings. One important gap in the application of GCaMP indicators is its postsynaptic physiological relevance. Correlation of synaptic GCaMP Ca²⁺ transients with postsynaptic currents simultaneously recorded by focal extracellular recording indicated that Ca²⁺ transients reported by GCaMPs were slow, and did not reflect immediate synaptic transmission. Rather, the kinetics of synaptic Ca²⁺ transients was temporally correlated with short-term synaptic plasticity such as facilitation and depression. The hyperexcitable ion channel mutations Sh and parabss¹ enhanced the synaptic Ca²⁺ transient amplitudes as well as depression. Type Is synapses of hyperexcitable mutations such as eag¹ Sh¹²⁰ and parabss¹ often displayed single stimulus pulse-evoked Ca²⁺ transients, which were induced by high frequency repetitive firing of action potentials. Such Ca²⁺ transients were correlated with supernumerary peaks of postsynaptic currents. Based on the slow kinetics and the correlation with short-term plasticity, I conclude that GCaMP Ca²⁺ signals better reflect the accumulation of cytosolic residual Ca²⁺. The spontaneous Ca²⁺ waves in larval motor neurons were well correlated with high frequency nerve action potentials, suggesting that accumulation of residual Ca²⁺ occurs in larval crawling. Overall, this study provided important information about the different excitability control and Ca²⁺ clearance mechanisms in different synapses, and examined how membrane excitability controls the influx and accumulation of synaptic cytosolic residual Ca2+, as indicated by GCaMPs. Further, by correlating synaptic Ca²⁺ dynamics with electrophysiology, this study also investigated how to interpret GCaMP Ca²⁺ signals in the context of facilitation and depression, establishing a basis for an integrated approach of studying short-term synaptic plasticity from complementary physiological signals.

The phenolamines octopamine and tyramine control, regulate, and modulate many physiological and behavioral processes in invertebrates. Vertebrates possess only small amounts of both substances, and thus, octopamine and tyramine, together with other biogenic amines, are referred to as “trace amines.” Biogenic amines evoke cellular responses by activating G-protein-coupled receptors. We have isolated a complementary DNA (cDNA) that encodes a biogenic amine receptor from the American cockroach Periplaneta americana, viz., Peatyr1, which shares high sequence similarity to members of the invertebrate tyramine-receptor family. The PeaTYR1 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with tyramine reduces adenylyl cyclase activity in a dose-dependent manner (EC50 350 nM). The inhibitory effect of tyramine is abolished by co-incubation with either yohimbine or chlorpromazine. Receptor expression has been investigated by reverse transcription polymerase chain reaction and immunocytochemistry. The mRNA is present in various tissues including brain, salivary glands, midgut, Malpighian tubules, and leg muscles. The effect of tyramine on salivary gland acinar cells has been investigated by intracellular recordings, which have revealed excitatory presynaptic actions of tyramine. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the cockroach.

Neurons perform complex computations, communications and precise transmissions of information in the form of action potentials (APs). The high level of heterogeneity and complexity at all levels of organization within a neuron and the functional requirement of highly permeable cell membranes leave neurons exposed to damage when energy levels are insufficient for the active maintenance of ionic gradients. When energy is limiting the ionic gradient across a neuron’s cell membrane risks being dissipated which can have dire consequences. Other researchers have advocated “generalized channel arrest” and/or “spike arrest” as a means of reducing the neuronal permeability allowing neurons to adjust the demands placed on their electrogenic pumps to lower levels of energy supply. I investigated the consequences of hypoxia on the propagation of a train of APs down the length of a fast conducting axon capable of transmitting APs at very high frequencies. Under normoxic conditions I found that APs show conduction velocities and instantaneous frequencies nearly double that of neurons experiencing energy limiting hypoxic conditions. I show that hypoxia affects AP conduction differently for different lengths of axon and for APs of different instantaneous frequencies. Action potentials of high instantaneous frequency in branching lengths of axon within ganglia were delayed more significantly than those in non-branching lengths contained within the connective and fail preferentially in branching axon. I found that octopamine attenuates the effects of hypoxia on AP propagation for the branching length of axon but has no effect on the non-branching length of axon. Additionally, for energetically stable cells, application of the anti-diabetic medication metformin or the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 resulted in a reduced performance similar to that seen in neurons experiencing energetic stress. Furthermore both metformin and ZD7288 affect the shape of individual APs within an AP train as well as the original temporal sequence of the AP train, which encodes behaviourally relevant information. I propose that the reduced performance observed in an energetically compromised cell represents an adaptive mechanism employed by neurons in order to maintain the integrity of their highly heterogeneous and complex organization during periods of reduced energy supply. / Thesis (Master, Biology) -- Queen's University, 2011-10-07 14:41:46.972

Flies are highly visually guided animals. In this thesis, I have used hoverflies as a model for studying motion vision. Flies process motion vision in three visual ganglia: the lamina, the medulla, and the lobula complex. In the posterior part of lobula complex, there are around 60 lobula plate tangential cells (LPTCs). Most of LPTCs have large receptive fields where the local direction sensitivity suggests that they function as matched filters to specific types of optic flow. LPTCs connect to descending or neck motor neurons that control wing and head movements, respectively. Therefore, in this thesis I have focused on the electrophysiological responses of LPTCs to gain understanding of visual behaviors in flies. The elementary motion detector (EMD) is a model that can explain the formation of local motion sensitivity. However, responses to higher order motion, where the direction of luminance change is uncorrelated with the direction of movement, cannot be predicted by classic EMDs. Nevertheless, behavior shows that flies can see and track bars with higher order motion cues. I showed (Paper I) that several LPTCs also respond to higher order motion. Many insects, including flies, release octopamine during flight. Therefore, adding octopamine receptor agonists can mimic physical activity. Our study (Paper II) investigated the effect of octopamine on three adaptation components. We found that the contrast gain reduction showed a frequency dependent increase after octopamine stimulation. Since the contrast gain is non-directional, it is likely presynaptic to the LPTC. We therefore believe that octopamine acts on the delay filter in the EMD. In the third paper we describe a novel LPTC. The centrifugal stationary inhibited flicker excited (cSIFE) is excited by flicker and inhibited by stationary patterns. Neither of these responses can be predicted by EMD models. Therefore, we provide a new type of motion detector that can explain cSIFE’s responses (Paper III). During bar tracking, self-generated optic flow may counteract the steering effect by inducing a contradictory optomotor response. Behavior shows that during bar fixation, flies ignore background optic flow. Our study (Paper IV) focus on the different receptive fields of two LPTCs, and relate these to the bar fixation behavior. In the neuron with a small and fronto-dorsal receptive field, we find a higher correlation with bar motion than with background motion. In contrast, the neuron with a larger receptive field shows a higher correlation with background motion.

Caffeine consistently enhances endurance performance in temperate environmental conditions, while far less research has examined its ergogenic and physiological effects during prolonged exercise in the heat. Despite the performance benefit of an acute caffeine doses being less pronounced in regular caffeine users versus those not habituated to the drug, few studies have examined the influence of a prolonged period of controlled caffeine intake on endurance performance. The endogenous trace amine octopamine is purported to possess stimulant-like properties and influence fat metabolism, although no study has examined these effects in humans. The aim of this thesis was to further characterise the performance and physiological effects of caffeine during prolonged exercise, while elucidating a potential ergogenic role for octopamine. The first two studies investigated the ergogenic and thermoregulatory effects of low to moderate caffeine doses during prolonged cycle exercise in the heat. Chapter 4 demonstrated that 3 mg kg-1 caffeine, administered either as a single or split-dose (2 x 1.5 mg kg-1) before exercise, improved endurance performance without influencing thermoregulation during prolonged exercise at a fixed work-rate. Dividing the caffeine bolus appeared to confer an additional performance benefit, suggesting repeated low dose may potentiate the efficiency of the same total caffeine dose under these conditions. Chapter 5 demonstrated that a 6 mg kg-1 caffeine dose improved endurance cycle performance without differentially influencing thermoregulation than placebo. The level of habituation to caffeine influences the ergogenic effect of an acute dose, yet previous studies have employed sub-chronic supplementation protocols. Chapter 6 investigated the effect of a twenty-eight day supplementation period on endurance cycle performance. Habituation to caffeine attenuated the ergogenic effect of an acute caffeine dose, without any change in circulating caffeine, substrate oxidation or hormonal concentrations. In chapter 7 the performance and metabolic effects of octopamine was investigated. Octopamine supplementation did not influence performance, hormonal concentrations or substrate oxidation, likely due to low serum concentrations of the drug.

L’acarien Varroa destructor est l’un des principaux responsables de l’effondrement des colonies d’abeilles domestique Apis mellifera. L’arsenal thérapeutique disponible pour lutter contre ce parasite ubiquiste apparait insuffisant à ce jour. Dans le cadre de cette thèse, la démarche de drug design, généralement utilisée en santé humaine, a été appliquée pour le développement de nouveaux médicaments vétérinaire à usage varroacide. Les travaux de cette thèse se sont focalisés sur deux cibles du système nerveux : l’acétylcholinestérase et les récepteurs à l’octopamine. Ces deux cibles ont déjà montré leur intérêt varroacide, notamment au travers des médicaments contenant du coumaphos ou de l’amitraze. Concernant l’acétylcholinestérase, un criblage fait avec le modèle 3D de l’enzyme, construit par homologie de séquences, a permis d’identifier deux composés de la chimiothèque du CERMN. Nous avons également exploré le potentiel varroacide d’acaricides de la famille des carbamates, démontrant l’intérêt du pirimicarbe comme varroacide. Concernant l’octopamine, l’étude de quatre dérivés de l’amitraze a montré l’intérêt de l’un d’entre eux. Un criblage par similarité de structure avec ce dérivé a mis en exergue une molécule issue de la chimiothèque du CERMN. Toutes les molécules pointées par ces travaux de thèse montrent de bons résultats lors de tests in vitro ou in vivo. Toutefois, afin de minimiser le risque pour l’abeille et maximiser l’efficacité anti-varroa, ces leads doivent être optimisés avant d’être ajouté à l’arsenal des médicaments varroacides. / The mite Varroa destructor is one of the main contributors to the collapse of honey bee colonies Apis mellifera. The therapeutic arsenal available against this ubiquitous parasite appears insufficient to date. In this thesis, the drug design approach, generally used in human health, was applied to the development of new varroacide veterinary drugs.The works of this thesis focused on two nervous system targets: acetylcholinesterase and octopamine receptors. These two targets have already shown their varroacide interest, especially through drugs containing coumaphos or amitraz. Regarding acetylcholinesterase, a screening made on the 3D model of the enzyme, built by sequence homology, allowed to identify two compounds from the CERMN compound library. We also explored the varroacide potential of carbamate acaricides, demonstrating the interest of pirimicarb as a varroacide. Regarding octopamine, the study of four derivatives of amitraz has shown the interest of one of them. Structural similarity screening with this derivative highlighted one hit from the CERMN compound library. All molecules pointed out by these thesis works show good results during in vitro or in vivo tests. However, to minimize the risk to honey bees and maximize their anti-varroa efficiency, these leads need to be optimized before being added to the arsenal of varroacide drugs.

The biogenic amines serotonin (5HT), dopamine (DA), and octopamine (OA) have been indicated in the regulation of behaviors, including aggression. The flesh fly, Sarcophaga crassipalpis, was used to investigate ontogenetic and circadian changes in amines and aggression. Heads of male flies were analyzed for amine content using high performance liquid chromatography with electrochemical detection (HPLC-ECD) at 3 time points on each of 4 consecutive days in 2 light cycles, 12:12 LD and 15:9 LD. Both DA and OA levels decreased with age. Light-cycle dependent differences were observed for all amines in overall levels and patterns of change throughout the day. A behavioral assay quantified interactive and aggressive behaviors at three time points in the light period for 2 age groups. The daily changes in behavioral profiles differed dependent on age. Correlations from these data can be made between changes in amine levels and time of day, photoperiod, age, and aggressive state.

Bugula neritina is a sessile marine bryozoan with a pelagic larval stage. Larvae frequently settle on boat hulls, facilitating the introduction of B. neritina to bays and estuaries worldwide. Adrenergic agonists, such as the vertebrate hormone noradrenaline, inhibit larval settlement in a variety of marine invertebrate species, including B. neritina. Light also inhibits B. neritina larval settlement, yet the underlying mechanisms by which light and adrenergic compounds exert their effects on larvae are not well understood. Octopamine is considered the invertebrate analog of noradrenaline, and may be an endogenous hormone involved in larval settlement pathways. I observed the effects of the adrenergic agonist noradrenaline and the adrenergic antagonist phentolamine on larval settlement, and found that high concentrations of noradrenaline increased larval mortality, inhibited larval attachment, and increased larval swimming behavior. High concentrations of phentolamine also increased larval mortality, but increased larval attachment and decreased larval swimming behavior. I used fluorescent labeling and microscopy to localize sensory system components, and found that larvae possess adrenergic-like receptors, as well as tyrosine hydroxylase-like and octopamine-like immunoreactivity. I also exposed larvae to phentolamine in both dark and light conditions, and found that light significantly inhibited larval attachment, but phentolamine blocked those inhibitory effects. These results suggest that B. neritina larvae possess adrenergic-like receptors, which serve as the binding sites for noradrenaline and phentolamine. These are likely octopamine receptors, and octopamine may be one endogenous compound involved in controlling larval phototaxis and settlement behavior. Light may increase octopamine production, thereby stimulating cilial activity, extending swimming behavior, and preventing larvae from attaching to a substrate. This research sheds light on previously unknown sensory mechanisms in B. neritina larvae, and may aid in the development of new biofouling control strategies.

No description available.

Evaluating our environment by deciding what is beneficial or harmful, pleasant or punishing is a part of our daily lives. Seeking pleasure and avoiding pain is a common trait all mobile organisms exhibit and understanding how rewarding stimuli are represented in the brain remains a major goal of neuroscience. Studying reward learning in the fruit fly, Drosophila melanogaster has enabled us to better understand the complex neural circuit mechanisms involved in reward processing in the brain. By conditioning flies with sugars of differing nutritional properties, we determined that flies trained with sweet but non-nutritive sugars formed robust short-term memory (STM), but not long-term memory (LTM). However, flies conditioned with a sweet and nutritious sugar or a sweet non-nutritious sugar supplemented with a tasteless nutritious compound, formed robust 24 hour LTM. These findings led us to propose a model of parallel reinforcement pathways for appetitive olfactory conditioning in the fly, in which both sweet taste and nutrient value contribute to appetitive long-term memory. We followed this line of research by examining the neural circuitry in the fly brain that represents these parallel reward pathways. We found that the biogenic amine octopamine (OA) only represents the reinforcing effects of sweet taste. Stimulation of OA neurons could replace sugar in olfactory conditioning to form appetitive STM. Surprisingly, implanting memory with OA was dependent on dopamine (DA) signaling, which although being long associated with reward in mammals, was previously linked with punishment in flies. We found that OA-reinforced memory functions through the α-adrenergic OAMB receptor in a novel subset of rewarding DA neurons that innervate the mushroom body (MB). The rewarding population of DA neurons is required for sweet and nutrient reinforced memory suggesting they may integrate both signals to drive appetitive LTM formation. In addition, OA implanted memory requires concurrent modulation of negatively reinforcing DA neurons through the β-adrenergic OCTβ2R receptor. These data provide a new layered reward model in Drosophila in which OA modulates distinct populations of both positive and negative coding DA neurons. Therefore, the reinforcement system in flies is more similar to that of mammals than previously thought.