Substance P content in cultured neonatal rat vagal sensory neurons: the effect of nerve growth factor

Abstract

To begin to study the factors regulating the synthesis and release of substance P (SP) in the sensory vagus nerve, cultures of neonatal rat nodose ganglia were developed. In microexplant cultures, obtained from small fragments of nodose ganglia, SP was present in low amounts: after 3 weeks,141 ± 36pg per well, 10 ganglia equivalents per well. To enhance neuron survival, nodose ganglia were enzymatically dissociated using neutral protease. Estimated survival at 5 days was 20–30%, with 800–1200 surviving neurons per plated ganglion, and decreased slowly thereafter. Specific SP immunostaining was present in 10–20% of neurons, mostly of small diameter (18–22 μm). SP content was low for 5 days then rose progressively after 14 days to 80–150 pg per plated ganglion. The addition of nerve growth factor (NGF, 100 ng/ml) to the culture medium did not alter neuron survival. However, SP content was doubled in the presence of NGF, or fell rapidly to one-half control levels following its withdrawal: e.g. following 12 days in culture with NGF1185 ± 176pg/well vs NGF withdrawn day 8–12,592 ± 118pg/well, mean±S.D., P < 0.01. Somatostatin, present in one-sixth the amount of SP, was unaltered by NGF. In subsequent studies, plating of neurons onto previously dissociated rat atriacytes increased survival by 50% but did not alter SP content per surviving neurons. These studies demonstrate that SP is present in dissociated cultures of rat vagal sensory neurons; the quantities and estimated net synthesis rate correspond to previous observations in vivo. The studies also demonstrate that SP content but not neuron survival are regulated by NGF in nodose ganglion neurons. This model may prove valuable for the study of SP and other sensory neuropeptides in this important class of visceral afferent neurons.