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Biogenic amines are important messenger substances in the central nervous system and in peripheral organs of vertebrates and of invertebrates. The honeybee, Apis mellifera, is excellently suited to uncover the functions of biogenic amines in behaviour, because it has an extensive behavioural repertoire, with a number of biogenic amine receptors characterised in this insect. In the honeybee, the biogenic amines dopamine, octopamine, serotonin and tyramine modulate neuronal functions in various ways. Dopamine and serotonin are present in high concentrations in the bee brain, whereas octopamine and tyramine are less abundant. Octopamine is a key molecule for the control of honeybee behaviour. It generally has an arousing effect and leads to higher sensitivity for sensory inputs, better learning performance and increased foraging behaviour. Tyramine has been suggested to act antagonistically to octopamine, but only few experimental data are available for this amine. Dopamine and serotonin often have antagonistic or inhibitory effects as compared to octopamine. Biogenic amines bind to membrane receptors that primarily belong to the large gene-family of GTP-binding (G) protein coupled receptors. Receptor activation leads to transient changes in concentrations of intracellular second messengers such as cAMP, IP3 and/or Ca2+. Although several biogenic amine receptors from the honeybee have been cloned and characterised more recently, many genes still remain to be identified. The availability of the completely sequenced genome of Apis mellifera will contribute substantially to closing this gap. In this review, we will discuss the present knowledge on how biogenic amines and their receptor-mediated cellular responses modulate different behaviours of honeybees including learning processes and division of labour.

In Drosophila, eight insulin-like peptides (DILP1-8) are produced and secreted in different locations. They regulate many aspects of development and physiology, such as organism growth, metabolic homeostasis, reproduction, stress resistance and life span. DILP2, 3 and 5 are mainly produced by a cluster of median neurosecretory cells in the brain known as insulin producing cells (IPCs). Here we showed that IPCs are under tight regulation of two G-protein coupled receptors (GPCRs), serotonin receptor 5-HT1A and octopamine receptor OAMB. Genetic manipulations of these two receptors in IPCs affected transcription levels of DILPs, hence altered feeding, carbohydrate levels, and resistance to stress (Paper I and II). Moreover, we showed that the insulin receptor (dInR) is strongly expressed in leucokininergic neurons (LK neurons), and selectively regulates growth of around 300 neuropeptidergic neurons expressing the bHLH transcription factor DIMMED. Overexpression of dInR in DIMM-positive neurons led to substantial neuronal growth, including cell body size, golgi apparatus and nuclear size, while knockdown of dInR had the opposite effect (Paper III). Manipulations of components in the insulin signaling pathway in LK neurons resulted in the similar cell size phenotypes. Furthermore, dInR regulated size scaling of DIMM-postive neurons is nutrient-dependent and partially requires the presence of DIMM (Paper III). Finally, we investigated the roles of DILPs (2, 3, 5 and 7) and LK neurons in regulation of feeding and diuresis at the adult stage (Paper IV).  In summary, we have identified two more regulators for IPC activity and demonstrated developmental roles of  DILPs and dInR in regulating neuronal size. Moreover, DILPs regulate water homeostasis together with a diuretic hormone leucokinin and as a consequence affects feeding behavior. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press. Paper 4: Manuscript.</p>

abstract: Animals must learn to ignore stimuli that are irrelevant to survival, which is a process referred to as ‘latent inhibition’. This process has been shown to be genetically heritable (Latshaw JS, Mazade R, Sinakevitch I, Mustard JA, Gadau J, Smith BH (submitted)). The locus containing the AmTYR1 gene has been shown through quantitative trait loci mapping to be linked to strong latent inhibition in honey bees. The Smith lab has been able to show a correlation between learning and the AmTYR1 receptor gene through pharmacological inhibition of the receptor. In order to further confirm this finding, experiments were designed to test how honey bees learn with this receptor knocked out. Here this G-protein coupled receptor for the biogenic amine tyramine is implemented as an important factor underlying latent inhibition in honey bees. It is shown that double-stranded RNA (dsRNA) and Dicer-substrate small interfering RNA (dsiRNA) that are targeted to disrupt the tyramine receptors specifically affects latent inhibition but not excitatory associative conditioning. The results therefore identify a distinct reinforcement pathway for latent inhibition in insects. / Dissertation/Thesis / Masters Thesis Biology 2017

<p>One of the outstanding goals of neuroscience is to understand how neural circuits are assembled to produce context appropriate behavior. In an ever changing environment, it is critical for animals to be able to flexibly respond to different stimuli to optimize their behavioral responses accordingly. Oviposition, or the process of choosing where to lay eggs, is an important behavior for egg-laying animals, yet the neural mechanisms of this behavior are still not completely understood. Here, we use the genetically tractable organism, Drosophila melanogaster, to investigate how the brain decides which substrates are best for egg deposition. We show that flies prefer to lay eggs away from UV light and that induction egg-laying correlates with increased movement away from UV. Both egg-laying and movement aversion of UV are mediated through R7 photoreceptors, but only movement aversion is mediated through Dm8 amacrine neurons. We then identify octopaminergic neurons as being potential modulators of egg-laying output. Collectively, this work reveals new insights into the neural mechanisms that govern Drosophila egg-laying behavior.</p> / Dissertation

Circadian rhythms are ubiquitous among organisms, influencing a wide array of physiological processes and behaviors including aggression. While many neurophysiological mechanisms are involved in the regulation of aggressive behaviors, relatively few studies have investigated the underlying components involved in the interplay between circadian rhythms and aggression. Spiders are an ideal model system for studying circadian regulation of aggression as they are ecologically both predators and prey. Recent studies have revealed a nocturnal orb- weaving spider Larinioides cornutus exhibits a diel and circadian rhythm in anti-predator behavior (i.e. boldness) that can be manipulated by administration of octopamine (OA) and serotonin (5- HT). Dosing of OA increases boldness of an individual while 5-HT decreases boldness levels. Thus, it appears the serotonergic and octopaminergic system are playing a key role in the daily fluctuations of boldness. This study took a holistic approach to investigate OA and 5-HT levels of head tissue and hemolymph (i.e. blood) as well as the genes involved in synthesis, signaling, and degradation of these monoamines throughout the day (0100, 0700, 1300, and 1900 hours) using HPLC-ED and RNA-sequencing. Although endogenous and circulating levels of OA did not significantly fluctuate, putative transcripts involved in synthesis and signaling did increase in relative expression levels at dusk when L. cornutus begins to actively forage for prey. Endogenous and circulating levels of 5-HT also did not significantly change at the four different time points, but clear patterns of upregulation of 5-HT synthesis enzymes as well as some receptor transcripts were upregulated during the day when L. cornutus would be mostly inactive in its retreat. Lastly, monoamine oxidase, a major catabolic enzyme of monoamines in vertebrates and some invertebrates, was identified in L. cornutus and exhibited substrate specificity for OA compared to 5-HT. Together with the higher enzymatic activity at mid-day compared to dusk, MAO appears to be playing a significant role in regulating the OA and 5-HT signaling in L. cornutus. In conclusion, these results allow a unique preliminary perspective on how OA and 5-HT are influencing the diel shifts in aggression-related behaviors in an ecologically dynamic arthropod.

Movement is controlled by neuronal central pattern generator (CPG) networks that are segmentally organised in organisms across the animal kingdom. The precise role of neuromodulators in the function, development and, particularly, the maintenance of these circuits is currently unresolved. This study investigates the effects of chronically altered signalling of tyramine and/or octopamine, two well established neuromodulators, in Drosophila larval locomotion. It shows that tyramine reduces crawling speed in larvae, whereas octopamine increases speed up to a physiological maximum. Changes in crawling speed are mediated by modulating stride duration, whilst stride length remains constant. These two neuromodulators also affect segmental muscle contraction and relaxation rates, indicative that the effects on crawling speed are likely to be at least partially due to modulatory effects on muscle physiology. Muscle recordings from muscle M6 in two adjacent segments, during fictive forward locomotion show that stride duration is influenced by a variable time delay between segmental CPG outputs. Frequency and duration of individual segmental outputs, by contrast, remains constant. The behavioural and electrophysiological data suggest, therefore, that the segmental locomotor CPG outputs remain constant in response to chronically altered neuromodulatory signalling. This study also identified a close spatial proximity of motor neuronal dendritic branches and putatively octopaminergic and/or tyraminergic synaptic terminal varicosities in the ventral nerve cord (VNC) neuropil. Moreover, manipulation of a putatively octopaminergic and/or tyraminergic subpopulation of interneurons, located in anterior brain regions, is sufficient to induce a similar, albeit smaller, larval crawling deficit. This indicates that the effects of locomotion may be induced in the central nervous system. This is confirmed in identified motor neurons as chronic changes in octopaminergic and/or tyraminergic signalling increase the frequency of bursting of action potential firing. In addition, the synaptic current amplitudes are substantially reduced in both ventral and dorsal muscle- innervating motor neurons, indicative of an effect to presynaptic excitation. In contrast, the function of neuromuscular junction remains largely unchanged. Taken together, this data shows that neuromodulation is sufficient to alter the output of a relatively small group of neurons, that comprise the locomotor CPG. The site of action of these modulators is, however, likely to be diverse.

Monoamine oxidase (MAO) is a mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of monoamines in a wide array of organisms. While the enzyme monoamine oxidase has been studied extensively in its role in moderating behavior in mammals, there is a paucity of research investigating this role in invertebrates, where the latter utilizes this enzyme in a major pathway to degrade monoamines. There is especially a dismal lack of information on how MAO influences activity in invertebrates, particularly in account of the circadian cycle. Previous studies revealed MAO degrades serotonin and norepinephrine in arachnids, but did not investigate other critically important compounds like octopamine. Larinioides cornutus is a species of orb-weaving spider that exhibits diel fluctuations in behavior, specifically levels of aggression. The monoamines octopamine and serotonin have been shown to influence aggressive behaviors in L. cornutus, thus this species was used to investigate if MAO is a potential site of regulation throughout the day. Not only did gene expression of MAO orthologs and MAO activity fluctuate at different times of day, but the enzymatic activity was substrate-specific producing a higher level of degradation of octopamine as compared to serotonin in vitro. This study further supports evidence that MAO has an active role in monoamine inactivation in invertebrates and provides a first look at how MAO ultimately may be regulating behavior in an invertebrate.

How are neuromodulatory networks organised to adapt sensory discrimination for different contexts? I hypothesised that neurons within a sensory circuit express different neuromodulatory receptors for differential modulation. Here I aimed to use the simple and genetically amenable Drosophila larval Mushroom Body (MB) calyx, a higher order processing area involved in learned odour discrimination, as a model to map octopamine (OA) neuromodulatory circuitry. I first identified olfactory projection neurons (PNs), a GABAergic feedback neuron and cholinergic extrinsic neurons as putative postsynaptic partners to OA neurons in the MB calyx using GFP reconstitution across synaptic partners. Next, I used novel EGFP-tagged OA receptors generated from recombination-mediated cassette exchange with MiMIC insertions in receptor genes to visualise endogenous expression patterns of OA receptors. Most notably, this is the first report of α2-adrenergic-like OA receptor localisation in any insect. For the first time, I showed that the α1-adrenergic-like OAMB localised to PN presynaptic terminals in the calyx; while Octβ1R localised diffusely in the calyx, resembling the innervation pattern of MB neuron dendrites. I detected EGFP-tagged Octα2R and Octβ2R in some PN cell bodies but not in neuron terminals - suggesting that Octα2R and Octβ2R may be expressed in some PNs, provided the misfolded fusion proteins are retained in the cell bodies of the neurons they are normally expressed in. Furthermore, I found that Octα2R and GABAAR fusion proteins localised to OA cell bodies but not to neuronal terminals, suggesting that OA neurons are subjected to inhibition, again given that these are not artefacts of the fusion proteins. To obtain tools to study OA modulation in the larval calyx, I then confirmed the expression patterns of driver lines that more specifically labelled calyx-innervating OA and extrinsic neurons, and tested the efficacy of three OAMB receptor knockdown lines. This initial attempt of mapping OA receptors, while subjected to further verification and development, is consistent with my hypothesis that a single neuromodulatory source can regulate multiple neuronal types in the same circuit through the distribution of different types of neuromodulatory receptors. This provides a new perspective in how the anatomical organisation of neuromodulation within a sensory network may translate to flexible outputs.