Establishment of a method to evaluate the plasticity and maturation of the dopaminergic nerve terminal

• Autor(a) principal: Neto, Diogo António Bessa
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/5671
• topic: Câmaras Microfluídicas; Cromatografia Líquida de Alta Performance (Hplc); Dopamina (da); Mesencéfalo; Sinapse; Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas

The modulatory midbrain dopamine (DA)ergic system is involved in important functions such as control of voluntary movement, reinforcement, learning and state of mind. Moreover, it is directly associated with some of most studied neuropathologies like Parkinson’s disease, Huntington’s disease and schizophrenia, and also drug addiction. Together, these functions/associations make the DAergic system an appealing field of study in neuroscience. Neurotransmission occurs predominantly through chemical synapses and is already well studied for glutamatergic hippocampal neurons. Nevertheless, DAergic transmission is still poorly investigated and many questions remain about how DA is transmitted. Embrionary ventral midbrain cultures are poor in DAergic neurons and current culture techniques inadequate to conduct axonal studies in these impoverished cultures. Thus, we tried to implement a microfluidic culture device that allows the physical and fluidic axonal isolation from somatodendritic ‘contamination’, with the help of glial cell line-derived neurotrophic factor supplementation to improve the DAergic survival. We showed that cells were maintained viable in microfluidic chambers for at least 14 days in culture, however, the number of DAergic axons was still low, not only due to the culture method limitations but also due to more broad problems associated with the cell cultures which occurred at same time. In addition, we optimized the detection of monoamine neurotransmitters and their metabolites present in embrionary ventral midbrain cultures by high-performance liquid chromatography coupled to electrochemical detection. In our cultures, only DA was detected, and was drastically reduced when cells were stimulated by the DAergic toxin 1-methyl-4-phenylpyridinium (MPP+). While the number of labelled cells for tyrosine hydroxylase was not affected by MPP+, at the concentrations tested.