Anelosimus studiosus is a socially polyphenic spider. Individuals can be classified as social/tolerant or solitary/aggressive. These behavioral differences are associated with considerable variation in social structure. Here, we begin to examine the physiological differences that may underlie the behavioral dimorphism in this species and possible implications for the evolution of sociality. Octopamine is a neurotransmitter that has been found to elevate aggression in invertebrates. Serotonin has been shown, in some cases, to interact antagonistically with octopamine. We used High Pressure Liquid Chromatography with Electrochemical Detection to quantify levels of these neurochemicals among adult females from social (multi-female) and solitary (single-female) webs in east Tennessee. A subset of spiders was scored for individual social tendency. We found that higher octopamine levels are associated with a greater degree of aggression and intolerance, both at the individual level and the population level, while higher levels of serotonin are found in multi-female colonies and social individuals.

À partir des ovocytes de la palourde Spisula solidissima, un ADNc codant un récepteur nommé Spi-OAR a été cloné et séquencé. Une analyse de la séquence en acides aminés a indiqué que ce nouveau récepteur possède une forte similarité avec les récepteurs β-adrénergiques et les récepteurs à octopamine. En effet, il est étroitement lié à la classe des récepteurs à octopamine « β-adrénergique-like » couplés à une protéine Gs. L’ADNc de Spi-OAR a été introduit dans un vecteur d'expression (pCEP4) et un épitope reconnaissable par un anticorps commercial a été ajouté au segment N-terminal. Cette construction a été transfectée dans des cellules hôtes (HEK 293) et des études d’immunofluorescence ont montré une expression efficace du récepteur au niveau membranaire. Également, des mesures d'AMPc pour les cellules exprimant Spi-OAR ont révélé une augmentation de ce messager secondaire lors de l'ajout de l'octopamine, et dans une moindre mesure, la tyramine, tandis que la dopamine, la sérotonine et l'histamine n’ont engendré aucun effet. Une légère activité constitutive de ce récepteur dans les cellules hôtes a été observée. De plus, une analyse RT-PCR avec des oligonucléotides spécifiques a révélé l'ARNm de Spi-OAR non seulement dans les ovocytes, mais aussi dans les gonades, le cœur, les muscles adducteurs, les branchies et les ganglions suggérant que ce récepteur soit exprimé de façon ubiquitaire dans divers tissus et dans différents stades embryonnaires chez la palourde. En outre, des études avec des ovocytes isolés n'ont montré aucun effet de l’octopamine sur la réactivation méiotique. Des études éventuelles pourront finalement confirmer le rôle fonctionnel de Spi-OAR. / A cDNA encoding for an octopamine receptor named Spi-OAR was cloned and sequenced from the surf clam Spisula solidissima oocytes. An analysis of its predicted amino acid sequence showed a high degree of similarity with β-adrenergic and octopamine receptors. This receptor qualifies as a novel receptor closely related to the proposed class of insect octopamine « β-adrenergic–like » receptors coupled to Gs protein. This cDNA was introduced into an expression vector (pCEP4), with an added N-terminal FLAG tag sequence, and transfected in host cells (HEK 293). Immunofluorescence studies showed expression of the receptor with a proper localization to the plasma membrane. Measurements of cAMP in transfected cells revealed that addition of octopamine, and to a lower extent, tyramine induced a rise in cAMP while dopamine, serotonine and histamine had no effect. Overexpression of Spi-OAR in mammalian cells induced slight constitutive increase of cAMP. An RT-PCR analysis with specific oligonucleotides revealed the presence of the receptor mRNA not only in oocytes but also in whole gonads, heart, adductor muscle, gills and ganglia suggesting that this receptor is likely ubiquitously expressed. Expression of Spi-OAR was also detected at different embryonic stages. Despite the demonstrated expression of Spi-OAR in oocytes, octopamine had no effect on meiotic reinitiation. Further studies will examine the function of Spi-OAR.

Em abelhas eussociais o controle das condições internas do ninho é importante para a sobrevivência da colônia e um dos principais mecanismos para controle da temperatura colonial é a escolha do local de nidificação, como ocos de troncos de árvores, tal qual ocorre nas abelhas sem ferrão Melipona quadrifasciata, sistema biológico deste estudo. Além de mecanismos comportamentais, em Hymenoptera são também conhecidos mecanismos fisiológicos de termorregulação (termogênese) como a ocorrência de ciclos fúteis (descritos para mamangavas Bombus) e o tremor muscular. A termogênese em abelhas ocorre principalmente durante incubação da cria, atividades sociais e aquecimento pré-vôo. As aminas biogênicas desempenham um amplo espectro de funções em abelhas, e é sugerido que o sistema noradrenérgico/adrenérico de vertebrados é funcional e estruturalmente equivalente ao sistema octopaminérgico/tiraminérgico de insetos. O emprego de bloqueador beta-adrenérgico em abelhas pode levar a hipotermia, emergindo a hipótese de que haveria envolvimento de uma via octopaminérgica na energética e termogênese de abelhas. Os três principais aspectos da termorregulação focados neste trabalho são a termorregulação colonial, a energética individual e mecanismos de termogênese. Com relação à termorregulação colonial, o objetivo principal foi descrevê-la em duas regiões do ninho (discos de cria e potes de alimento), por meio de registro de séries temporais de temperatura e umidade relativa, e estudo da atividade externa de vôo. Foi detectada ritmicidade nas séries temporais de colônias abertas, com influência das periodicidades da temperatura ambiente, umidade relativa e luminosidade, sobre as séries temporais registradas dentro da colônia. As séries temporais de atividade de vôo apresentavam periodicidades similares às ambientais, mas também eram relacionadas às da cria. Em colônias fechadas, as séries temporais dos discos de cria apresentavam periodicidades próximas a 24h e 12h, sugerindo aspecto endógeno da regulação de temperatura e umidade próximo à cria. Também, objetivou-se caracterizar o efeito do tipo de iluminação e de diferentes temperaturas. Foram estudados, por meio de respirometria intermitente, os efeitos do ciclo claro/escuro a 28°C (fotoperíodo normal, fotoperíodo invertido, claro e escuro constante) sobre a taxa metabólica (TM), e os efeitos de diferentes temperaturas ambientais (10 a 40°C) sobre a TM e quociente respiratório (QR). Os diferentes ciclos de claro/escuro afetaram a TM, sendo observados, em fotoperíodo normal e claro constante valores de TM baixos entre 19h e 5h. Em fotoperíodo invertido, valores diminuídos foram registrados entre 6h e 18h, sugerindo um ritmo exógeno em resposta a mudanças fotoperiódicas. Entre 7h e 17h59, no claro, ressalta-se que não há diferenças estatisticamente significativas na TM. A temperatura tem um efeito significativo, com valores diminuídos a 35 e 40°C e mais ainda a 10°C. Entre 15 e 30°C não há diferenças significativas. Na terceira parte do trabalho objetivou-se verificar o efeito tempo-dependente de bloqueadores beta-adrenérgicos e octopaminérgicos (Alprenolol e Mianserina) sobre a TM e temperatura torácica de forrageadoras e verificar se a octopamina e methoprene (análogo de hormônio juvenil) compensam os efeitos dos bloqueadores. Também foi analisado se há alterações dos substratos energéticos catabolizados, considerando o QR e a atividade enzimática (Vmax de hexoquinase, trealase, HOAD e glicogênio fosforilase). Com o emprego dos bloqueadores, foram detectadas alterações no QR e na atividade das quatro enzimas. A diminuição da temperatura torácica obtida em forrageadoras tratadas com Alprenolol sugere o envolvimento de vias octopaminérgicas na termogênese em M. quadrifasciata; todavia, não se sabe se tais efeitos são decorrentes de interferências na produção de calor e/ou um subproduto da diminuição da TM. No nível colonial, ninhos tratados com os mesmos bloqueadores parecem apresentar periodicidades de séries temporais mais similares à da umidade relativa do ambiente e menos à da temperatura ambiente, sugerindo menor resposta às variações de temperatura. Os resultados não provam que há mecanismos de termogênese sem tremor muscular em abelhas, envolvendo octopamina e hormônio juvenil, mas indicam um possível envolvimento destas substâncias na taxa metabólica e oxidação de diferentes substratos energéticos. Sob uma visão comparativa, pode-se apontar semelhanças entre efeitos de modificações em vias octopaminérgicas/tiraminérgicas de insetos e noradrenérgicas/tiraminérgicas de vertebrados, quando à energética e termogênese. Os resultados não provam tal afirmação, mas apontam evidências do papel da via octopaminérgica na taxa metabólica e temperatura torácica de forrageadoras. / In eusocial insects, such as stingless bees, the control of nest conditions and maintenance of the colonial microclimate are important to brood incubation, development of eggs, larvae and pupae, and survival of the colony. One of the main thermoregulatory mechanisms to control nest temperature is the microhabitat selection to build the nest, such as tree holes, characteristic of the stingless bee Melipona quadrifasciata, the biological system chosen for this study. In addition to the behavioral mechanisms involved in thermoregulation, physiological mechanisms underlying thermogenesis are also found in Hymenoptera, such as futile cycles (in bumblebees) and shivering thermogenesis. Thermogenesis in bees is detected mainly during brood incubation, social interactions and pre-flight warming; additional possible physiological mechanisms have not yet been investigated with enough detail. Biogenic amines, especially octopamine, play important physiological roles in bees. Although octopamine and norepinephrine are chemically not identical, it appears that octopaminergic systems of invertebrates and noradrenergic systems of vertebrates are homologous. Oral treatment with beta-blockers can cause hypothermia, leading to the hypothesis that an octopaminergic pathway involved in bee energetics and thermogenesis might exist. The main aspects investigated in the present study are the colonial thermoregulation, the energetics of individual bees, and the mechanisms associated with thermogenesis in M. quadrifasciata. With regards to the colonial thermoregulation, temperature and humidity, time series were recorded inside and outside the nest, in two colonial compartments, i.e., brood and pots. In addition, flight activity was also recorded. Flight activity time series presented similar periodicities in both ambient and brood series. In closed colonies, maintained under constant conditions, the brood time series presented periodicities close to 24h and 12h, suggesting endogenous aspects and rhythm. Another goal of this study was to characterizing the effects of the light-dark cycles and ambient temperatures. This was achieved using intermittent respirometry, and the effects of the light-dark cycles at 28°C (normal photoperiod, inverted photoperiod, constant light and constant dark) on metabolic rate (MR), and the effects of different temperatures (10 40°C) on MR and respiratory quotient (RQ) were measured. The different light-dark cycles affected the MR under normal photoperiod and constant light. Two \"platforms,\" with low MR values were detected between 19h and 5h. When the photoperiod was inverted, lower values were recorded in the dark phase, between 6h and 18h, resulting in an inverted pattern of MR, thus suggesting an exogenous response to photoperiodic changes. Between 7h and 17h59, in the light phase, metabolic rate did not change significantly. The temperature has a significant effect on MR and RQ of the foragers, and reduced values at 10, 35 and 40°C were detected. Between 15 and 30°C no significant differences were detected. In the third part of this work the aim was to verify the time-dependent effects of beta-blockers (Alprenolol and Mianserina) on MR and thoracic temperature of foragers, and to verify whether octopamine and methoprene (analogous to juvenile hormone) compensate the effects of beta-blockers. Alterations of substrates oxidation, considering the RQ and the activity (Vmax) of specific enzymes, such as hexokinase, trehalase, HOAD and glycogen phosphorylase, were also investigated. Treatments with blockers caused alterations in the RQ and in the enzyme activities of hexokinase, HOAD and glycogen phosphorylase. The reduced values of thoracic temperature in foragers treated with Alprenolol suggests the involvement of octopaminergic pathways in thermogenesis; however, it is not yet known if such effects are due to interferences in the heat production and/or represent a by-product of a reduced metabolic rate. At the colonial level, nests treated with blockers presented colonial time series periodicities more similar to the ambient humidity series than to the ambient temperature series, suggesting therefore that the responses to ambient temperature are reduced. These results do not prove that mechanisms of nonshivering thermogenesis are present in bees, involving both octopamin and juvenile hormone, but they can indicate possible involvements of these substances in metabolic rate, energetics and fuel utilization. Under a comparative approach, one can suggest similarities between the effects caused by modifications in the noradrenergic and octopaminergic pathways on the energetics and thermogenesis of M. quadrifasciata. The results, however, do not prove such hypothesis, but they suggest an octopaminergic influence on both metabolic rate and thoracic temperature of M. quadrifasciata foragers.

The importance of social behaviour is evident in human society, but there are both costs and benefits associated with cooperation and sociality throughout the animal kingdom. At what point do the benefits outweigh the costs, and when do selective pressures favour sociality and colonization over solitude and independence? To investigate these questions, we have focused on an anomalous species of spider, Anelosimus studiosus, also known now as the northern social spider. Throughout its broad range, A. studiosus is solitary and aggressive, but recently, colonies of cooperative and social individuals have been observed at northern latitudes. This leads to two research questions: 1) what characteristics differentiate the two variants behaviourally, and, 2) how are they different physiologically? Colonies and individuals were collected from multiple populations throughout the Tennessee River watershed area and maintained in a laboratory environment for quantitative and qualitative assessment of behavioural traits as well as specific neurochemical analysis by high performance liquid chromatography with electrochemical detection. After classifying individuals as social or aggressive, I looked at the influence of factors such as age, reproductive state, nutritional state, and time of day on behaviour and neurophysiology. I found correlations between social behaviours and serotonin, aggressive behaviours and octopamine (invertebrate counterpart of norepinephrine), and several other compounds associated with an increase or decrease in aggression. These studies combine techniques from multiple disciplines to contribute to the greater understanding of the proximate control of social and aggressive behaviours as well as factors influencing the evolution of sociality.

À partir des ovocytes de la palourde Spisula solidissima, un ADNc codant un récepteur nommé Spi-OAR a été cloné et séquencé. Une analyse de la séquence en acides aminés a indiqué que ce nouveau récepteur possède une forte similarité avec les récepteurs β-adrénergiques et les récepteurs à octopamine. En effet, il est étroitement lié à la classe des récepteurs à octopamine « β-adrénergique-like » couplés à une protéine Gs. L’ADNc de Spi-OAR a été introduit dans un vecteur d'expression (pCEP4) et un épitope reconnaissable par un anticorps commercial a été ajouté au segment N-terminal. Cette construction a été transfectée dans des cellules hôtes (HEK 293) et des études d’immunofluorescence ont montré une expression efficace du récepteur au niveau membranaire. Également, des mesures d'AMPc pour les cellules exprimant Spi-OAR ont révélé une augmentation de ce messager secondaire lors de l'ajout de l'octopamine, et dans une moindre mesure, la tyramine, tandis que la dopamine, la sérotonine et l'histamine n’ont engendré aucun effet. Une légère activité constitutive de ce récepteur dans les cellules hôtes a été observée. De plus, une analyse RT-PCR avec des oligonucléotides spécifiques a révélé l'ARNm de Spi-OAR non seulement dans les ovocytes, mais aussi dans les gonades, le cœur, les muscles adducteurs, les branchies et les ganglions suggérant que ce récepteur soit exprimé de façon ubiquitaire dans divers tissus et dans différents stades embryonnaires chez la palourde. En outre, des études avec des ovocytes isolés n'ont montré aucun effet de l’octopamine sur la réactivation méiotique. Des études éventuelles pourront finalement confirmer le rôle fonctionnel de Spi-OAR. / A cDNA encoding for an octopamine receptor named Spi-OAR was cloned and sequenced from the surf clam Spisula solidissima oocytes. An analysis of its predicted amino acid sequence showed a high degree of similarity with β-adrenergic and octopamine receptors. This receptor qualifies as a novel receptor closely related to the proposed class of insect octopamine « β-adrenergic–like » receptors coupled to Gs protein. This cDNA was introduced into an expression vector (pCEP4), with an added N-terminal FLAG tag sequence, and transfected in host cells (HEK 293). Immunofluorescence studies showed expression of the receptor with a proper localization to the plasma membrane. Measurements of cAMP in transfected cells revealed that addition of octopamine, and to a lower extent, tyramine induced a rise in cAMP while dopamine, serotonine and histamine had no effect. Overexpression of Spi-OAR in mammalian cells induced slight constitutive increase of cAMP. An RT-PCR analysis with specific oligonucleotides revealed the presence of the receptor mRNA not only in oocytes but also in whole gonads, heart, adductor muscle, gills and ganglia suggesting that this receptor is likely ubiquitously expressed. Expression of Spi-OAR was also detected at different embryonic stages. Despite the demonstrated expression of Spi-OAR in oocytes, octopamine had no effect on meiotic reinitiation. Further studies will examine the function of Spi-OAR.

Connectomics – the study of neural connectivity – is primarily concerned with the mapping and characterisation of wired synaptic links; however, it is well established that long-distance chemical signalling via extrasynaptic volume transmission is also critical to brain function. As these interactions are not visible in the physical structure of the nervous system, current approaches to connectomics are unable to capture them. This work addresses the problem of missing extrasynaptic interactions by demonstrating for the first time that whole-animal volume transmission networks can be mapped from gene expression and ligand-receptor interaction data, and analysed as part of the connectome. Complete networks are presented for the monoamine systems of Caenorhabditis elegans, along with a representative sample of selected neuropeptide systems. A network analysis of the synaptic (wired) and extrasynaptic (wireless) connectomes is presented which reveals complex topological properties, including extrasynaptic rich-club organisation with interconnected hubs distinct from those in the synaptic and gap junction networks, and highly significant multilink motifs pinpointing locations in the network where aminergic and neuropeptide signalling is likely to modulate synaptic activity. Thus, the neuronal connectome can be modelled as a multiplex network with synaptic, gap junction, and neuromodulatory layers representing inter-neuronal interactions with different dynamics and polarity. This represents a prototype for understanding how extrasynaptic signalling can be integrated into connectomics research, and provides a novel dataset for the development of multilayer network algorithms.

Em abelhas eussociais o controle das condições internas do ninho é importante para a sobrevivência da colônia e um dos principais mecanismos para controle da temperatura colonial é a escolha do local de nidificação, como ocos de troncos de árvores, tal qual ocorre nas abelhas sem ferrão Melipona quadrifasciata, sistema biológico deste estudo. Além de mecanismos comportamentais, em Hymenoptera são também conhecidos mecanismos fisiológicos de termorregulação (termogênese) como a ocorrência de ciclos fúteis (descritos para mamangavas Bombus) e o tremor muscular. A termogênese em abelhas ocorre principalmente durante incubação da cria, atividades sociais e aquecimento pré-vôo. As aminas biogênicas desempenham um amplo espectro de funções em abelhas, e é sugerido que o sistema noradrenérgico/adrenérico de vertebrados é funcional e estruturalmente equivalente ao sistema octopaminérgico/tiraminérgico de insetos. O emprego de bloqueador beta-adrenérgico em abelhas pode levar a hipotermia, emergindo a hipótese de que haveria envolvimento de uma via octopaminérgica na energética e termogênese de abelhas. Os três principais aspectos da termorregulação focados neste trabalho são a termorregulação colonial, a energética individual e mecanismos de termogênese. Com relação à termorregulação colonial, o objetivo principal foi descrevê-la em duas regiões do ninho (discos de cria e potes de alimento), por meio de registro de séries temporais de temperatura e umidade relativa, e estudo da atividade externa de vôo. Foi detectada ritmicidade nas séries temporais de colônias abertas, com influência das periodicidades da temperatura ambiente, umidade relativa e luminosidade, sobre as séries temporais registradas dentro da colônia. As séries temporais de atividade de vôo apresentavam periodicidades similares às ambientais, mas também eram relacionadas às da cria. Em colônias fechadas, as séries temporais dos discos de cria apresentavam periodicidades próximas a 24h e 12h, sugerindo aspecto endógeno da regulação de temperatura e umidade próximo à cria. Também, objetivou-se caracterizar o efeito do tipo de iluminação e de diferentes temperaturas. Foram estudados, por meio de respirometria intermitente, os efeitos do ciclo claro/escuro a 28°C (fotoperíodo normal, fotoperíodo invertido, claro e escuro constante) sobre a taxa metabólica (TM), e os efeitos de diferentes temperaturas ambientais (10 a 40°C) sobre a TM e quociente respiratório (QR). Os diferentes ciclos de claro/escuro afetaram a TM, sendo observados, em fotoperíodo normal e claro constante valores de TM baixos entre 19h e 5h. Em fotoperíodo invertido, valores diminuídos foram registrados entre 6h e 18h, sugerindo um ritmo exógeno em resposta a mudanças fotoperiódicas. Entre 7h e 17h59, no claro, ressalta-se que não há diferenças estatisticamente significativas na TM. A temperatura tem um efeito significativo, com valores diminuídos a 35 e 40°C e mais ainda a 10°C. Entre 15 e 30°C não há diferenças significativas. Na terceira parte do trabalho objetivou-se verificar o efeito tempo-dependente de bloqueadores beta-adrenérgicos e octopaminérgicos (Alprenolol e Mianserina) sobre a TM e temperatura torácica de forrageadoras e verificar se a octopamina e methoprene (análogo de hormônio juvenil) compensam os efeitos dos bloqueadores. Também foi analisado se há alterações dos substratos energéticos catabolizados, considerando o QR e a atividade enzimática (Vmax de hexoquinase, trealase, HOAD e glicogênio fosforilase). Com o emprego dos bloqueadores, foram detectadas alterações no QR e na atividade das quatro enzimas. A diminuição da temperatura torácica obtida em forrageadoras tratadas com Alprenolol sugere o envolvimento de vias octopaminérgicas na termogênese em M. quadrifasciata; todavia, não se sabe se tais efeitos são decorrentes de interferências na produção de calor e/ou um subproduto da diminuição da TM. No nível colonial, ninhos tratados com os mesmos bloqueadores parecem apresentar periodicidades de séries temporais mais similares à da umidade relativa do ambiente e menos à da temperatura ambiente, sugerindo menor resposta às variações de temperatura. Os resultados não provam que há mecanismos de termogênese sem tremor muscular em abelhas, envolvendo octopamina e hormônio juvenil, mas indicam um possível envolvimento destas substâncias na taxa metabólica e oxidação de diferentes substratos energéticos. Sob uma visão comparativa, pode-se apontar semelhanças entre efeitos de modificações em vias octopaminérgicas/tiraminérgicas de insetos e noradrenérgicas/tiraminérgicas de vertebrados, quando à energética e termogênese. Os resultados não provam tal afirmação, mas apontam evidências do papel da via octopaminérgica na taxa metabólica e temperatura torácica de forrageadoras. / In eusocial insects, such as stingless bees, the control of nest conditions and maintenance of the colonial microclimate are important to brood incubation, development of eggs, larvae and pupae, and survival of the colony. One of the main thermoregulatory mechanisms to control nest temperature is the microhabitat selection to build the nest, such as tree holes, characteristic of the stingless bee Melipona quadrifasciata, the biological system chosen for this study. In addition to the behavioral mechanisms involved in thermoregulation, physiological mechanisms underlying thermogenesis are also found in Hymenoptera, such as futile cycles (in bumblebees) and shivering thermogenesis. Thermogenesis in bees is detected mainly during brood incubation, social interactions and pre-flight warming; additional possible physiological mechanisms have not yet been investigated with enough detail. Biogenic amines, especially octopamine, play important physiological roles in bees. Although octopamine and norepinephrine are chemically not identical, it appears that octopaminergic systems of invertebrates and noradrenergic systems of vertebrates are homologous. Oral treatment with beta-blockers can cause hypothermia, leading to the hypothesis that an octopaminergic pathway involved in bee energetics and thermogenesis might exist. The main aspects investigated in the present study are the colonial thermoregulation, the energetics of individual bees, and the mechanisms associated with thermogenesis in M. quadrifasciata. With regards to the colonial thermoregulation, temperature and humidity, time series were recorded inside and outside the nest, in two colonial compartments, i.e., brood and pots. In addition, flight activity was also recorded. Flight activity time series presented similar periodicities in both ambient and brood series. In closed colonies, maintained under constant conditions, the brood time series presented periodicities close to 24h and 12h, suggesting endogenous aspects and rhythm. Another goal of this study was to characterizing the effects of the light-dark cycles and ambient temperatures. This was achieved using intermittent respirometry, and the effects of the light-dark cycles at 28°C (normal photoperiod, inverted photoperiod, constant light and constant dark) on metabolic rate (MR), and the effects of different temperatures (10 40°C) on MR and respiratory quotient (RQ) were measured. The different light-dark cycles affected the MR under normal photoperiod and constant light. Two \"platforms,\" with low MR values were detected between 19h and 5h. When the photoperiod was inverted, lower values were recorded in the dark phase, between 6h and 18h, resulting in an inverted pattern of MR, thus suggesting an exogenous response to photoperiodic changes. Between 7h and 17h59, in the light phase, metabolic rate did not change significantly. The temperature has a significant effect on MR and RQ of the foragers, and reduced values at 10, 35 and 40°C were detected. Between 15 and 30°C no significant differences were detected. In the third part of this work the aim was to verify the time-dependent effects of beta-blockers (Alprenolol and Mianserina) on MR and thoracic temperature of foragers, and to verify whether octopamine and methoprene (analogous to juvenile hormone) compensate the effects of beta-blockers. Alterations of substrates oxidation, considering the RQ and the activity (Vmax) of specific enzymes, such as hexokinase, trehalase, HOAD and glycogen phosphorylase, were also investigated. Treatments with blockers caused alterations in the RQ and in the enzyme activities of hexokinase, HOAD and glycogen phosphorylase. The reduced values of thoracic temperature in foragers treated with Alprenolol suggests the involvement of octopaminergic pathways in thermogenesis; however, it is not yet known if such effects are due to interferences in the heat production and/or represent a by-product of a reduced metabolic rate. At the colonial level, nests treated with blockers presented colonial time series periodicities more similar to the ambient humidity series than to the ambient temperature series, suggesting therefore that the responses to ambient temperature are reduced. These results do not prove that mechanisms of nonshivering thermogenesis are present in bees, involving both octopamin and juvenile hormone, but they can indicate possible involvements of these substances in metabolic rate, energetics and fuel utilization. Under a comparative approach, one can suggest similarities between the effects caused by modifications in the noradrenergic and octopaminergic pathways on the energetics and thermogenesis of M. quadrifasciata. The results, however, do not prove such hypothesis, but they suggest an octopaminergic influence on both metabolic rate and thoracic temperature of M. quadrifasciata foragers.

Ectothermic animals are susceptible to temperature changes such as cold shock with seasons. To survive through a cold shock, ectotherms have developed unique strategies. My interest is focusing on the physiological function of during cold shock and prolonged cold exposure in the fruit fly (Drosophila melanogaster) and crayfish (Procambarus clarkii). I used Drosophila melanogaster as a model system to investigate cardiac function in response to modulators (serotonin, acetylcholine, octopamine, dopamine and a cocktail of modulators) in acute cold shock and chronic cold shock conditions as possible mechanism to regulate heart rate in the cold. To examine if the dampened heart rate in the cold could still be enhanced by modulators or calcium loading, modulators and light-sensitive channelrhodopsin proteins were utilized to stimulate the heart. This light induced cardiac activation increased heart rate in all conditions, and potentially can be used for cardiac therapy in mammals. Also, the acute and chronic cold conditioned heart showed responsiveness to the above mention modulators. In examining how synaptic transmission is influenced by acute and chronic cold, the crayfish neuromuscular junction was used as a model. This is a good model as there are high and low output synapses to be investigated. The low output neuromuscular junction was enhanced in response to acute cold. The high output nmj increased in synaptic response to acute cold. In addressing chronic cold conditions, the nmj were physiologically assayed in their response to acute warm changes as well as influence of serotonin and octopamine. In chronic cold condition, the synaptic output was varied in enhanced and dampened responses to an acute warm environment. These junctions were enhanced in their synaptic output by serotonin and octopamine (100nM). In assessing, by HPLC assay, octopamine concentration increased in chronic cold crayfish. This suggests compensation in synaptic transmission in cold acclimation possibility via endocrine responses.

Synaptic plasticity—the ability of a synapse to change—is fundamental to basic brain function and behavioral adaptation. Studying the mechanisms of synaptic plasticity benefits our understanding of the formation of neuronal connections and circuitry, which has great implications in the field of learning and memory and the studies of numerous human diseases. The Drosophila larval neuromuscular junction (NMJ) system is a powerful system for studying synaptic plasticity. The NMJ consists of at least two different types of motorneurons innervating the body wall muscles. Type I motorneurons controls muscle contraction using glutamate as the neurotransmitter, while type II are modulatory neurons that contain octopamine. Octopamine is a potent modulator of behavior in invertebrates. Nevertheless, its function at the synapse is poorly understood. In my thesis research, I investigated the role of octopamine in synaptic plasticity using the Drosophila NMJ system. Preliminary observations indicate that increased larval locomotion during starvation results in an increase of filopodia-like structures at type II terminals. These structures, which we termed as “synaptopods” in our previous studies, contain synaptic proteins and can mature into type II synapses. I demonstrated that this outgrowth of type II terminals is dependent on activity and octopamine. Mutations and genetic manipulations affecting the production of octopamine decrease synaptopods, whereas increase of type II activity or exogenous application of octopamine increase synaptopods. Interestingly, I found that the type II octopaminergic neurons have an absolute dependence on activity for their innervation of the muscles. Blocking activity in these neurons throughout development results in no type II synapses at the NMJ, whereas blocking activity after the formation of synapses results in gradual degradation of type II terminals. Next, I examined the autoregulatory mechanism underlying the octopamine-induced synaptic growth in octopaminergic neurons. I discovered that this positive-feedback mechanism depends on an octopamine autoreceptor, Octß2R. This receptor in turn activates a cAMP- and CREB-dependent pathway that is required in the octopamine-induction of synaptopods. Furthermore, I demonstrated that this octopaminergic autoregulatory mechanism is necessary for the larva to properly increase its locomotor activity during starvation. Thirdly, I investigated the possibility that type II innervation might regulate type I synaptic growth through octopamine. We found that ablation, blocking of type II activity, or the absence of octopamine results in reduced type I outgrowth, and this paracrine signaling is mediated by Octß2R which is also present in type I motorneurons. Lastly, the function of another octopamine receptor, Octß1R, was examined. In contrast to Octß2R, Octß1R is inhibitory to synaptic growth. I demonstrated that the inhibitory effect of this receptor is likely accomplished through the inhibitory G-protein Goα. Similar to Octß2R, Octß1R also regulates the synaptic growth of both type I and type II motorneurons in a cell-autonomous manner. The inhibitory function of this receptor potentially breaks the positive feedback loop mediated by Octß2R, allowing the animal to reset its neurons when the environment is favorable. In summary, the research presented in this thesis has unraveled both autoregulatory and paracrine mechanisms in which octopamine modulates synaptic and behavior plasticity through excitatory and inhibitory receptors.

Anxiety disorders are the most prevalent psychiatric conditions within primary care, affecting up to 29% of people across their lifetime. Generalized Anxiety disorder (GAD) is frequently comorbid with Major Depressive Disorder (MDD), resulting in greater functional impairment. Gut microbiota have been shown to modulate brain chemistry and function, possibly also playing a role in the genesis of anxiety. Bacteria are also able to produce, or interact with the host metabolism of neuroactive substances, including classical neurotransmitters and trace amines, like octopamine, which although found in trace concentrations in the mammalian brain, can affect CNS function. Specifically, trace amines can affect catecholamine release, reuptake and biosynthesis, and modulate dopamine and serotonin metabolism. We investigated whether microbiota from patients with GAD with no signs of immune activation can alter behaviour in gnotobiotic mice and whether this is accompanied by changes in metabolites within the gastrointestinal tract. Germ-free NIH Swiss mice (n=35) were colonized with microbiota from either a GAD patient (n=18) with severe anxiety, comorbid depression, and low serum and fecal octopamine, or an age and sex-matched healthy control (HC) (n=17). Three weeks post- colonization, mouse behaviour was assessed by standard psychometric tests. Emotionality z-scores were calculated to provide a robust integrated behavioural assessment. Microbiota profiles were assessed by 16S rRNA based Illumina, fecal β-defensin-3 level was measured by ELISA. After sacrifice, mouse brain BDNF and GDNF expression was assessed by immunofluorescence, and gene expression in the hippocampus, amygdala, and olfactory bulbs was assessed by Nanostring. Stool and cecum metabolites were measured in all colonized mice by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). There were no differences in fecal β-defensin levels between mice colonized with GAD microbiota as compared to mice colonized with HC microbiota. However, GAD mice exhibited greater anxiety and depressive-like behavior compared to HC mice in the digging and tail suspensions tests. Behavioural z-scoring across all six standard psychometric tests showed a significant increase in group emotionality score means of GAD-colonized mice compared to HC-colonized mice. Mice colonized with microbiota from a GAD patient had distinct bacterial profiles from mice colonized with HC microbiota. Compared to HC mice, GAD mice had lower levels of dopamine, octopamine and acetylcholine in cecum contents. Furthermore, GAD mice had higher expression of BDNF in the amygdala, lower expression of BDNF in the hippocampus, and lower expression of GDNF in the midbrain. GAD mice also had lower expression of CCR2 in the hippocampus, higher Cnlp/CAMP in the amygdala and olfactory bulb, and higher Nfkb1 in the olfactory bulb compared to HC mice. Our results suggest that microbiota from a selected patient with GAD has the ability to induce anxiety and depressive-like behavior, by mechanisms independent of immune system, likely by altered production of biogenic amines and neurotransmitters. / Thesis / Master of Science (MSc)