Development of immunoreactivity for calcitonin gene-related peptide, substance p and glutamate in primary sensory neurons, and for serotonin in the spinal cord of fetal sheep

Abstract

In study we have described the ontogeny of immunoreactivity for calcitonin gene-related peptide, substance P and glutamate in primary sensory neurons, and for serotonin in the sacral spinal cord, of fetal sheep ( n = 37) from 56 to 140 days of gestation (term = 146 days). A few fine, varicose fibres immunoreactive for calcitonin gene-related peptide were present in Lissauer's tract, the dorsolateral funiculus and in laminae I and V in the dorsal horn of the spinal cord at 56–61 days of gestation. At this age, two groups of intensely staining immunoreactive cells were present in the motoneuron pool in laminae VIII and IX in the ventral horn of the spinal cord. By 77 days, immunoreactive fibres were also present in laminae II and X. With advancing gestational age, an increase in the intensity of staining was observed throughout the cord to term, with the exception of laminae VIII and IX, where a decrease was seen. Intense staining of cells in the motoneuron pool was evident until c. 128 days, after which time staining became very faint. Fine fibres immunoreactive for substance P were present in Lissauer's tract and lamina I of the spinal cord at 56–61 days of gestation. They were also present throughout laminae IV–VI and X as well as throughout the entire ventral horn. Immunoreactive fibres in lamina II were evident by 77 days. The staining increased in density but remained similar in distribution with increasing gestational age to term in the dorsal horn, but decreased markedly in the ventral horn. Cells immunoreactive for substance P were evident from 56 days, particularly on the border of laminae II and III, until late in gestation. Ultrastructural studies showed that axon terminals immunoreactive for calcitonin gene-related peptide and for substance P were present in lamina I by 61 days. Immunoreactivity for glutamate was evident at 83 days in dorsal root fibres and also in lamina I and II, where it was more prominent in cells than in fibres. At all ages examined, the dorsal horn stained more intensely than the ventral horn. Immunoreactivity for glutamate and neuropeptides appeared in the cells and fibres of dorsal root ganglia at 97–100 days. In the skin, immunoreactivity for calcitonin gene-related peptide and substance P was present at 85 days, some time after its appearance in the cord. Fibres immunoreactive for serotonin appeared in lamina I, at the neck of the dorsal horn and in the ventral horn at 83 days of gestation. Few immunoreactive fibres were seen at any stage during gestation in laminae II and III. Staining increased in laminae IV–X during gestation, but there was eventually a slight decrease in the ventral horn prior to term. These studies have shown that neurochemicals thought to be involved in the transmission and modification of somatosenory information in the spinal cord are present in fetal sheep from early in gestation, with neuropeptides appearing before glutamate and serotonin.