Steroidal regulation of coexisting neuronal messengers: Focus on double and triple immunolabeling procedures and on indirect evaluation of coexistence

The coexistence of peptides and glucocorticoid receptors (GR) in neuronal populations was analyzed by means of double immunocytochemical techniques, providing a morphological substrate for a regulatory action via glucocorticolds (GC) of peptide synthesis or post-translational events. Triple labelings of GR, corticotropin-releasing factor (CRF), and c-fos protein immunoreactivities at the paraventricular hypothalamic nucleus suggested an interaction between GR and c-fos in the impairment during stress of the GC feedback actions on CRF. These techniques enable direct visualization and quantitation at the light or fluorescence microscope level. The main advantages and drawbacks are pointed out. An indirect technique for quantitation of neuronal profiles using light microscopy, the occlusion method, is presented and compared with other techniques. Such tools have proved useful in analyzing the functional meaning of the coexistence of multiple peptides in discrete neuronal populations.