Region-specific immunolocalization of atrial natriuretic peptide in mixed fetal rat brain cell cultures

In adult rodent CNS, atrial natriuretic peptide (ANP) has been localized by immunolabeling and nucleic acid hybridization techniques primarily to hypothalamic neurons and, to a lesser extent, to neurons of the telencephalon and brainstem. In canine brain, ANP immunoreactivity has been reported in neocortical and brainstem astrocytes. Yet, in a recent study using fetal rat tissue, ANP was detected by radioimmunoassay in predominantly neuronal, but not glial, cultures. In the present study ANP was detected by radioimmunoassay on in vitro day 8 in media derived from fetal rat diencephalic and rhombencephalic, but not telencephalic, cultures and in cell homogenates from all 3 regions surveyed. Using indirect immunofluorescence less than one cell per 400x field stained for ANP in the telencephalic cultures and ANP immunopositivity colocalized exclusively to neurons by concomitant anti-neurofilament immunolabeling. In diencephalic monolayers, approximately 1-3 cells per 400x field were ANP-positive; although most of these cells were neurons, small numbers of ANP-positive astrocytes were also detected using anti-glial fibrillary acidic protein (GFAP) immunolabeling. ANP-positive cells were most numerous in rhombencephalic cultures (5-10 cells per 400x field); as in diencephalic cultures, ANP immunoreactivity colocalized to both neurons and astrocytes in this region. In diencephalon and rhombencephalon, less than 1% of all ANP-positive cells were astrocytes and less than 1% of GFAP-positive astrocytes exhibited immunoreactive ANP. In fetal brain, neuronal and astrocytic ANP may play a role in fluid and electrolyte homeostasis. Alternatively, fetal brain cell ANP may subserve entirely different functions (e.g. as trophic factors) as has been suggested for other neuropeptides in the developing nervous system.