Análise metabolômica do cérebro de abelhas (Apis mellifera) submetidas a ensaio de Reflexo de Extensão de Probóscide (REP)
Carregando...
Data
2016-02-23
Autores
Pratavieira, Marcel [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
As abelhas têm sido utilizadas como modelos robustos e influentes para o estudo de memória e aprendizagem, contribuindo para o melhor entendimento das bases da cognição. Nesse contexto, diferentes metabólitos foram caracterizados por desempenharem funções distintas no processo de aprendizagem e formação de memória em insetos. Considerando que pouco se sabe sobre os metabólitos em relação ao desenvolvimento das habilidades cognitivas em A. mellifera, ou mesmo em relação aos comportamentos reflexos (condicionados e/ou não condicionados), o presente estudo teve como objetivo a análise metabolômica do cérebro de abelhas submetidas ao ensaio comportamental de reflexo de extensão de probóscide (REP). Para isto, foi padronizada a técnica de análise metabolômica com o uso do sistema LC-ESI- MS e MSn, construindo-se inicialmente uma biblioteca de compostos característicos de cérebro de abelhas (neurotransmissores, aminoácidos livres, poliaminas, nucleotídeos, nucleosídeos, ácidos orgânicos, etc). Nesta primeira abordagem, dentre os 112 compostos da biblioteca, 48 foram identificados e quantificados; alguns destes compostos foram únicos para o grupo controle (cadaverina, espermina, glicose, uracila e n-acetil-L-glutamato 5-semialdeido), enquanto que outros foram únicos para o grupo REP (fenilalanina, betaína, espermidina, serina e creatina). Dentre os compostos identificados em ambos os grupos, apenas 5 compostos apresentaram diferenças quantitativas estatisticamente significantes (arginina, asparagina, guanosina monofosfato, putrescina e 4-guanitinobutanoato). Visando o estudo dos perfis metabólicos regionais (em cada região do cérebro), foi também padronizado o protocolo experimental utilizando-se a estratégia MALDI Spectral Imaging e o desenvolvimento de um método semi-quantitativo de metabólitos. Esta estratégia permitiu o mapeamento e o estudo da distribuição espacial dos metabólitos identificados em cortes de cérebro de abelhas, bem como uma melhor compreensão da distribuição dessas moléculas nas diferentes estruturas do cérebro e sua correlação com o comportamento ensaiado. As duas estratégias aplicadas mostraram-se complementares e fundamentais para a compreensão da cognição em abelhas. De modo geral, o ensaio de REP parece estimular intensa atividade cerebral, alto gasto energético, intensa sinalização química e a ativação de algumas cascatas metabólicas específicas, tais como a rota metabólica da prolina e arginina. Nos indivíduos do grupo REP a arginina provavelmente foi catabolizada nas sínteses de creatina, 4-guanidinobutanoato, putrescina e espermidina. Esses processos bioquímicos provavelmente foram importantes para coordenar o reconhecimento da molécula de sacarose, e associar esse odor com um comportamento reflexo para a extensão da probóscide (estímulo não condicionado).
The honeybee Apis mellifera has long served as an invertebrate model organism for learning and memory research, contributing to a better understanding of cognition bases. In this context, different metabolites (especially neurotransmitters) were characterized by play distinct roles in learning process and in memory formation in insects. Whereas little is known about the metabolites in relation to the development of cognitive skills in A. mellifera, the present study aims to perform a metabolomic analysis of the honeybee brains submitted to the behavioral test of proboscis extension reflex (PER). For this, has been standardized a metabolomic analysis technique through the use of LC-ESI-MS and MSn system. Initially a low molecular weight compounds library was created, containing characteristic compounds of bee brain (neurotransmitters, free amino acids, polyamines, nucleotides, nucleosides, organic acids, etc.). In this first approach, from the library of 112 compounds, 48 compounds were identified and quantified; some of these compounds were only identified in the control group (cadaverine, spermine, glucose, uracil and N-acetyl-L-glutamate 5- semialdehyde), while others were only identified in PER group (phenylalanine, betaine, spermidine, serine and creatine). Among the compounds identified in both groups, only five compounds showed statistically significant differences in quantitative results (arginine, asparagine, guanosine monophosphate, putrescine and 4-guanidinebutanoate). In order to study the metabolic profiles by regions (within each brain region) it was also standardized an experimental protocol using a novel semi-quantitative method of MALDI Spectral Imaging strategy. This strategy allowed the study and the mapping of the spatial distribution of metabolites identified in honeybee brain sections, as well as a better understanding of the distribution of molecules in the different brain structures and their correlation with the behavior tested. Both strategies applied proved to be complementary and essential to the understanding of cognition in bees. Overall, in this study the PER test seems to stimulate intense brain activity, high energy expenditure, intense chemical signaling and activation of some specific metabolic pathways, such the arginine and proline metabolic pathway. In individuals of PER group arginine was probably catabolized in the synthesis of creatine, 4-guanidinobutanoato, putrescine and spermidine. This biochemical process were probably important to coordinate the recognition of sucrose molecule and associate this odor with a reflex behavior for the proboscis extension (unconditioned stimulus).
The honeybee Apis mellifera has long served as an invertebrate model organism for learning and memory research, contributing to a better understanding of cognition bases. In this context, different metabolites (especially neurotransmitters) were characterized by play distinct roles in learning process and in memory formation in insects. Whereas little is known about the metabolites in relation to the development of cognitive skills in A. mellifera, the present study aims to perform a metabolomic analysis of the honeybee brains submitted to the behavioral test of proboscis extension reflex (PER). For this, has been standardized a metabolomic analysis technique through the use of LC-ESI-MS and MSn system. Initially a low molecular weight compounds library was created, containing characteristic compounds of bee brain (neurotransmitters, free amino acids, polyamines, nucleotides, nucleosides, organic acids, etc.). In this first approach, from the library of 112 compounds, 48 compounds were identified and quantified; some of these compounds were only identified in the control group (cadaverine, spermine, glucose, uracil and N-acetyl-L-glutamate 5- semialdehyde), while others were only identified in PER group (phenylalanine, betaine, spermidine, serine and creatine). Among the compounds identified in both groups, only five compounds showed statistically significant differences in quantitative results (arginine, asparagine, guanosine monophosphate, putrescine and 4-guanidinebutanoate). In order to study the metabolic profiles by regions (within each brain region) it was also standardized an experimental protocol using a novel semi-quantitative method of MALDI Spectral Imaging strategy. This strategy allowed the study and the mapping of the spatial distribution of metabolites identified in honeybee brain sections, as well as a better understanding of the distribution of molecules in the different brain structures and their correlation with the behavior tested. Both strategies applied proved to be complementary and essential to the understanding of cognition in bees. Overall, in this study the PER test seems to stimulate intense brain activity, high energy expenditure, intense chemical signaling and activation of some specific metabolic pathways, such the arginine and proline metabolic pathway. In individuals of PER group arginine was probably catabolized in the synthesis of creatine, 4-guanidinobutanoato, putrescine and spermidine. This biochemical process were probably important to coordinate the recognition of sucrose molecule and associate this odor with a reflex behavior for the proboscis extension (unconditioned stimulus).
Descrição
Palavras-chave
Metabolômica, Neurociência, Apis mellifera, Espectrometria de Massas, MALDI MSI, Neurobiologia, Metabolomics, Neuroscience, Mass spectrometry