Neuropeptide expression in cultures of adult sensory neurons: Modulation of substance P and calcitonin gene-related peptide levels by nerve growth factor

Abstract

In contrast to developing sensory neurons, the survival of adult rat dorsal root ganglion neurons in pure neuronal culture is not dependent on specific neurotrophic factors such as nerve growth factor or brain-derived neurotrophic factor [ Lindsay R.M. (1988) J. Neurosci. 8, 2394–2405]. In the present study we have examined possible modulatory effects of nerve growth factor on the neuropeptide content of sub-populations of adult rat dorsal root ganglion neurons in vitro. During the first 1–2 days in culture the neuropeptides substance P and calcitonin gene-related peptide could be detected by immunofluorescence staining in cultures grown in the presence or absence of nerve growth factor, but at longer times in nerve growth factor-deprived cultures there was loss of immunoreactive staining for both peptides. In the presence of nerve growth factor, however, the percentage of substance P- and calcitonin gene-related peptide-immunoreactive neurons remained relatively constant, for at least 14 days, at levels that were similar to the percentage of such peptide-containing neurons found in sections of adult rat dorsal root ganglia. Quantitation by radioimmunoassay of the levels of substance P and calcitonin gene-related peptide in cultures grown in the presence or absence of nerve growth factor agreed with the qualitative observations obtained by immunofluorescence: 10–15-fold higher levels of substance P and calcitonin gene-related peptide were found in cultures grown with nerve growth factor for 18 days, as compared to nerve growth factor-deprived cultures. In nerve growth factor-treated cultures increased levels of substance P and calcitonin gene-related peptide were observed within 3–6 days in vitro, and further steady increases in the levels of both peptides were found up to 18 days. A low basal level of both peptides could always be detected, even in the presence of an excess of antibodies to nerve growth factor. Up-regulation of the synthesis of substance P and calcitonin gene-related peptide did not depend on nerve growth factor being present at the initiation of the cultures, as elevated levels of both peptides could be induced in cultures even after up to 10 days' prior deprivation of nerve growth factor. Removal of nerve growth factor from the cultures resulted in reduced levels of peptide within 3 days. Taken together these results confirm and extend earlier studies in vivo which have suggested that nerve growth factor may play a dynamic role in the regulation of the levels of substance P in mature sensory neurons. In the present study, it has been established that the effects of nerve growth factor are not related to survival and that substance P is not the only neuropeptide whose level of synthesis is modulated by nerve growth factor.