RESUMO
Brain-derived neurotrophic factor (BDNF) has previously been shown by this and other laboratories to work in concert with dopamine (DA) to induce the dopaminergic phenotype in foetal rat and human cerebral cortex during specified sensitive developmental stages. In the present study this induction by BDNF/DA was found to be greatly amplified by adding forskolin (fsk: 10 microM) to the rat and human cerebral cortex cultures together with DA (10 microM) and BDNF (50 ng/ml). This amplification was 14-fold for human tissue and 2-fold for rat tissue treated over an 80% shorter period. Compared to treatment with BDNF alone, the additional fsk increased tyrosine hydroxylase-positive (TH+) cell numbers by 220-fold in the human and 26-fold in the rat tissue. Parallel reverse transcription-polymerase chain reaction (RT-PCR) measurement of TH mRNA showed substantial increases above control levels when BDNF/DA or BDNF/DA/fsk treatments were applied. Since fsk boosts intracellular levels of cyclic AMP (cAMP), its amplifying action when added together with BDNF/DA is likely to be due to interactions via the cAMP response element/cAMP response element binding protein (CRE/CREB) systems. This is discussed.
Assuntos
Córtex Cerebral/enzimologia , Colforsina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Neurônios/enzimologia , Transcrição Gênica/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Córtex Cerebral/embriologia , Primers do DNA , Dopamina/farmacologia , Embrião de Mamíferos , Feto , Humanos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina 3-Mono-Oxigenase/análiseRESUMO
The pattern of release of radioactive brain-derived neurotrophic factor ([125I]BDNF) from brain tissue was studied. Rat brain slices from cerebral cortex and synaptosomes from cerebral cortex and hippocampus were preloaded with [125I]BDNF. Depolarising stimulation by veratridine (final conc. 50 microM) and high KCl (final conc. 45 mM) caused a short-term, greatly enhanced depolarisation-induced release of [125I]BDNF during superfusion and batch protocol experiments. The results suggested that the evoked release was independent of the presence of extracellular calcium ions, but dependent on intracellular calcium ion stores, since the intracellular calcium ion chelator BAPTA-AM, but not the extracellular chelator EGTA abolished the high-potassium-induced [125I]BDNF release from synaptosomes. The release was blocked by tetrodotoxin (1 microM) when synaptosomes were stimulated by veratridine or potassium chloride. Short time-fraction (30 s) superfusion experiments showed that the [125I]BDNF release from synaptosomes appeared in two temporal phases.