Excitatory amino acids, monoamine, and nitric oxide synthase systems in organotypic cultures: biochemical and immunohistochemical analysis

The nigrostriatal and mesolimbic systems of the rat have been re-constructe d using the organotypic culture model, whereby neonatal brain tissue is gro wn in vitro for approximately one month. The nigrostriatal cultures consist ed of tissue from the substantia nigra, dorsal striatum and frontoparietal cortex; while the mesolimbic cultures included the ventral tegmental area, ventral striatum and cingulate cortex. The cultures were grown at 35 degrees C in normal atmosphere, using a tube- roller device placed in a cell incubator and changing the medium every 3-4 days. The in vitro development was evaluated with an inverted microscope eq uipped with a variable relief contrast function. Samples were taken directl y from the medium in the culture tube and analysed for several amino acids with HPLC. After a month the cultures were fixed and processed for immunohi stochemistry. High levels of glutamate and aspartate were observed every time the medium was changed, but the levels rapidly decreased reaching a steady state after approximately 24 h. A decrease in the levels was also observed along devel opment, reaching stable values (similar to 2 mu M and similar to 0.12 mu M for glutamate and aspartate, respectively) at approximately two weeks, but only when the cultures showed an apparently healthy development. The levels were approximately 10 times higher in deteriorating or apparently damaged cultures. Glutamine levels were in the mM range and remained stable along t he entire experiment. No differences were observed among nigrostriatal and mesolimbic cultures. Immunohistochemistry confirmed the impressions obtained from microscopic an d biochemical analysis along the in vitro development, revealing apparently healthy neuronal systems with characteristics similar to those observed in vivo, when tyrosine hydroxylase and nitric oxide synthase, markers for dop amine and nitric oxide containing neurons, respectively, were analysed. In the substantia nigra, nitric oxide synthase-positive networks surrounded ty rosine hydroxylase-positive neurons, while in the striatum nitric oxide syn thase dendrites were surrounded by tyrosine hydroxylase-positive nerve term inals, suggesting a reciprocal interaction among dopamine and nitric oxide containing neurons. Thus, the organotypic model appears to capture many of the neurochemical an d morphological features seen in vivo, providing a valuable model for study ing in detail the neurocircuitries of the brain.