RESUMEN
Neuronal plasticity allows an animal to respond to environmental changes by modulating its response to stimuli. In the honey bee (Apis mellifera), the biogenic amine octopamine plays a crucial role in appetitive odor learning, but little is known about how octopamine affects the brain. We investigated its effect in the antennal lobe, the first olfactory center in the brain, using calcium imaging to record background activity and odor responses before and after octopamine application. We show that octopamine increases background activity in olfactory output neurons, while reducing average calcium levels. Odor responses were modulated both upwards and downwards, with more odor response increases in glomeruli with negative or weak odor responses. Importantly, the octopamine effect was variable across glomeruli, odorants, odorant concentrations and animals, suggesting that the octopaminergic network is shaped by plasticity depending on an individual animal's history and possibly other factors. Using RNA interference, we show that the octopamine receptor AmOA1 (homolog of the Drosophila OAMB receptor) is involved in the octopamine effect. We propose a network model in which octopamine receptors are plastic in their density and located on a subpopulation of inhibitory neurons in a disinhibitory pathway. This would improve odor-coding of behaviorally relevant, previously experienced odors.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Antenas de Artrópodos/citología , Red Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Octopamina/farmacología , Animales , Abejas , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Fura-2/análogos & derivados , Fura-2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Odorantes , Análisis de Componente Principal , Receptores de Amina Biogénica/genética , Receptores de Amina Biogénica/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: In the antennal lobe, a dedicated olfactory center of the honeybee brain, odours are encoded as activity patterns of coding units, the so-called glomeruli. Optical imaging with calcium-sensitive dyes allows us to record these activity patterns and to gain insight into olfactory information processing in the brain. METHOD: We introduce ImageBee, a plugin for the data analysis platform KNIME. ImageBee provides a variety of tools for processing optical imaging data. The main algorithm behind ImageBee is a matrix factorisation approach. Motivated by a data-specific, non-negative mixture model, the algorithm aims to select the generating extreme vectors of a convex cone that contains the data. It approximates the movie matrix by non-negative combinations of the extreme vectors. These correspond to pure glomerular signals that are not mixed with neighbour signals. RESULTS: Evaluation shows that the proposed algorithm can identify the relevant biological signals on imaging data from the honeybee AL, as well as it can recover implanted source signals from artificial data. CONCLUSIONS: ImageBee enables automated data processing and visualisation for optical imaging data from the insect AL. The modular implementation for KNIME offers a flexible platform for data analysis projects, where modules can be rearranged or added depending on the particular application. AVAILABILITY: ImageBee can be installed via the KNIME update service. Installation instructions are available at http://tech.knime.org/imagebee-analysing-imaging-data-from-the-honeybee-brain.
Asunto(s)
Abejas/fisiología , Encéfalo/fisiología , Algoritmos , Animales , Abejas/anatomía & histología , Encéfalo/anatomía & histología , Calcio/metabolismo , Análisis por Conglomerados , Programas InformáticosRESUMEN
The vacuolar H(+)-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V(0) and V(1) subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V(1) components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V(1) translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Insectos/metabolismo , Glándulas Salivales/metabolismo , Serotonina/metabolismo , Transducción de Señal , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , AMP Cíclico/análogos & derivados , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Citoplasma/metabolismo , Dípteros , Activación Enzimática , Concentración de Iones de Hidrógeno , Potenciales de la Membrana , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas/farmacología , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Glándulas Salivales/efectos de los fármacos , Glándulas Salivales/enzimología , Factores de TiempoRESUMEN
Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H(+)-ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N-hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H(+) transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H(+) transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface.
Asunto(s)
Dípteros/enzimología , Glándulas Salivales/fisiología , Serotonina/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fluorescencia , Concentración de Iones de Hidrógeno , Proteínas de Insectos , Macrólidos/farmacología , Microelectrodos , Glándulas Salivales/citología , Glándulas Salivales/efectos de los fármacos , Serotonina/farmacología , Serotoninérgicos/farmacologíaRESUMEN
Reversible assembly of the V0V1 holoenzyme from V0 and V1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H+-ATPases (V-ATPases) in animal cells. In the blowfly (Calliphora vicina) salivary gland, V-ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the cAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that cAMP, Sp-adenosine-3',5'-cyclic monophosphorothioate, or forskolin, similar to serotonin, cause V-ATPase-dependent luminal acidification. In addition, V-ATPase-dependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V1 components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca2+, our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus.