Development of substance P receptors on rat motoneurons in vitro

Experiments were performed to examine the influence of interneuronal interactions on the expression of neurotransmitter receptors by developing mammalian CNS neurons. Receptors for the neuropeptide, substance P (SP), were assayed on embryonic rat motoneurons and other spinal cord neurons developing in vitro by the binding of 125I-SP to live neurons. Scatchard analysis showed the presence of high-affinity binding sites, and binding competition assays using SP, neurokinin A, or neurokinin B indicated that the high-affinity 125I-SP binding sites on these neurons were type NK1 tachykinin receptors, or SP receptors (SPRs). Neurons in the spinal cords of rats at Embryonic Day 14 displayed no SPRs. Cell-surface SPRs were detected on spinal cord neurons within 24 hr after they were placed in culture, however, and the level of 125I-SP binding increased for several days. SPRs were assayed on spinal motoneurons that had been identified by retrograde labeling with a fluorescent tracer, isolated in high purity by fluorescence-activated cell sorting (FACS), and maintained in culture. Motoneurons grown in isolation from other neurons developed SPRs in vitro along the same time course as neurons in heterogeneous spinal cord cultures. These results show that rat spinal motoneurons can express SPRs early in their development, and they suggest that the initial expression of SPRs by developing motoneurons does not require interaction with other neurons.