Localization and high-resolution imaging of cortical neurotransmitter compartments using confocal laser scanning...

This report compares the application of confocal laser scanning fluorescence microscopy with standard epifluorescence microscopy for the simultaneous localization of the neurotransmitters gamma-aminobutyric acid and glutamate in rat cerebral cortex. With this approach, sections of fixed rat brain are treated with primary antibodies against gamma-aminobutyric acid (rabbit-derived) and glutamate (mouse-derived), followed by treatment with fluorescein isothiocyanate-tagged donkey anti-rabbit and rhodamine-tagged goat anti-mouse secondary antibodies, respectively. The results demonstrate that images from immunofluorescence localizations with a confocal laser scanning microscope have superior resolution and contrast as a result of significant reductions of background flare caused by emission from out-of-focus structures in the field of view. The confocal microscope achieves this improved image quality by optically sectioning through a specimen at narrow planes of focus and then compiling a composite image of an object of interest. The composite image can be further enhanced by using various image processing options. The combined use of double immunofluorescence and confocal laser scanning microscopy provides an important means to simultaneously study the anatomical relationships of pre- and post-synaptic elements in a complex neural system.