Avaliação neuroquímica do sistema colinérgico de camundongos com o gene do transportador vesicular de acetilcolina (VAChT) modificado geneticamente

Abstract

The vesicular acetylcholine transporter (VACht) is responsible for acetylcholine (Ach) storage in synaptic vesicles, an important step for cholinergic transmission. Our goal was to study the role of VAChT in cholinergic tonus maintenance through the generation of mice presenting alterations in VAChT gene by homologous recombination.. One of used strategies was the insertion of a neomycin resistance cassette in untranslated 5 region of VAChT gene. This genetic modification generated mice HET HOM presenting reduced expression of VAChT (VAChT Knockdown). KD and KD present a 45% and 65% decrease in VAChT protein expression, respectively. Accumulation of not released Ach in brain of KD was observed and it is not related with increased expression or activity of ChAT (choline acetyltransferase) or CHT1 (High affinity choline transporter). Electrophysiological analysis at neuromuscular HET HOM and KD junction showed a decrease in quantal size distribution for both, KD specially in the second one. We produced mice in which VAChT gene was flanqued by LoxP sites to Flox/Flox generate VAChT mice. These mice were bred with mice expressing Cre enzyme in different regions of brain. The lineage generated would present deletion of VAChT Flox/Flox mice showed that gene in specific tissues. Our initial characterization of VAChT they presented decrease of 50% in VAChT mRNA expression, an increase of 100% in ChAT mRNA expression and an increase around 6 fold in brain Ach when compared to WT/WT VAChT animals. The amplitude, but not frequency, of miniature potentials Flox/Flox (MEEPs) in neuromuscular junction is increased in VAChT mice. However, Flox/Flox VAChT mice are phenotipically normals. Flox/WT Flox/Flox VAChT or VAChT animals were bred with mice expressing Cre Flox/Flox,cre+ under CAMKII promoter control. The generated VAChT are morphologicaly normals in birth, however, after five days of live they look smaller and Flox/Flox,cre+ not as active as the control littermates animals . Besides this, the VAChT mice present general muscular weakness, anormal development of spinal cord represented by a kyphosis and survive just 8 weeks. Another mice lineage was generated due to VAChT allele deletion: Flox/Del, cre+ Flox/Flox, cre+ VAChT . These mice present similar phenotype to VAChT mice; however this genotype present a reducted time life to approximately 20 days. Flox/Flox,cre- Flox/Del,cre- Even not shown Cre, VAChT and VAChT animals surprisely, present abolishment of VAChT in different regions of CNS. Besides this, ACh tissue Flox/Del, cre- WT/WT is 12 fold higher than in VAChT animals. content in VAChT Interestingly, comportamental and eletrophisiological studies, do not suggest alteration of VAChT, since the Cre- animals are phenotypically normals. Similarly to Flox/Del, cre+ Flox/Flox,cre- Flox/Del, cre- VAChT animals, VAChT and VAChT mice present VAChT mRNA, suggesting an unknown regulatory mechanism of VAChT expression. Our results confirm the important role of VAChT in cholinergic transmission through Ach releasing regulation and, in consequence, in maintaining normal physiological function of central and peripheral nervous system.