RESUMO
Choline acetyltransferase (ChAT, EC 2.3.1.6) synthesizes a neurotransmitter, acetylcholine in cholinergic neurons. ChAT is considered to be the most specific marker for cholinergic neurons. To obtain a better marker of the neurons, as the first step, we isolated a partial ChAT cDNA from the goldfish (Carassius auratus) brain by RT-PCR methods. The partial cDNA of the goldfish ChAT was composed of 718 nucleotides. The amino acid sequence of the goldfish ChAT is approximately 70% identical to those of mammalian and chicken ChAT. Northern blot analysis demonstrated that ChAT mRNA was expressed in the brain and the spinal cord of the goldfish, and much abundant in the spinal cord. In the spinal cord of the goldfish, ChAT-positive neurons were detected mainly in the ventral horn by in situ hybridization. In addition, fluorescence in situ hybridization combined with a retrograde labeling by using True Blue demonstrated ChAT mRNA positive neurons were exactly motoneurons. In the cord, putative presynaptic sympathetic neurons were also labeled.
Assuntos
Colina O-Acetiltransferase/genética , Carpa Dourada/metabolismo , Neurônios/enzimologia , RNA Mensageiro/metabolismo , Medula Espinal/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Colina O-Acetiltransferase/metabolismo , Clonagem Molecular , Carpa Dourada/genética , Hibridização In Situ , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
c-fos is an immediate early gene, and is rapidly and transiently induced in neurons of the central nervous system according to their activities. To investigate neuronal activities in the brain of the goldfish (Carassius auratus), we considered that expression of c-fos mRNA would be an available marker for the neuronal activities. Therefore, we firstly isolated a cDNA clone encoding c-Fos from the goldfish brain by RT-PCR and RACE methods. A full length cDNA of the goldfish c-fos was composed of 1044 bp open reading frame. The amino acid sequence of the goldfish c-Fos was approximately 56-90% identical to those of other teleostean fish c-Fos. Northern blot analysis showed that the expression of c-fos mRNA was rapidly and transiently induced in the brain of the goldfish by the intraperitoneal administration of kainic acid. We also showed that the identification of the c-fos mRNA expression site by in situ hybridization will be able to be used as an anatomical marker for the identification of the activated neuronal region in the goldfish brain.