Ultrastructural studies on peptides in the dorsal horn of the spinal cord—I. Co-existence of galanin with other peptides in primary afferents in normal rats

Abstract

The aim of the present study was to investigate galanin-like immunoreactivity in primary afferent terminals and its relationship to other neuropeptides in laminae I and II of the fourth and fifth lumbar segments of normal rat spinal cord using immunofluorescence and pre- and post-embedding electron-microscopic immunocytochemistry. Triple-immunofluorescence staining showed that galanin-like immunoreactivity co-localized with substance P- and calcitonin gene-related peptide-like immunoreactivities in many nerve fibres and terminals in laminae I and II of the dorsal horn. At the ultrastructural level, using pre-embedding immunocytochemistry, galanin-like immunoreactivity was found in type I glomeruli with an electron-dense central terminal containing many densely packed synaptic vesicles and several large dense-core vesicles. Both the cytoplasm and the core of the large vesicles were immunoreactive. In type II glomeruli with an electron-lucent central terminal and loosely packed synaptic vesicles the large dense-core vesicles and the cytoplasm were only weakly galanin-positive. Post-embedding immunocytochemistry revealed that galanin-like immunoreactivity co-existed with substance P- and calcitonin gene-related peptide-like immunoreactivities in many terminals and in individual large dense-core vesicles in lamina II. These terminals were considered to represent primary afferents, since there is evidence that calcitonin gene-related peptide in the dorsal horn only occurs in nerve endings originating in dorsal root ganglia. Evidence was also unexpectedly obtained for the occurrence of several other peptides in calcitonin generelated peptide-positive terminals, i.e. in presumably primary afferents. Thus galanin-like immunoreactivity sometimes also co-localized with cholecystokinin- and neuropeptide tyrosine-like immunoreactivities in calcitonin gene-related peptide-immunoreactive terminals and in some large dense-core vesicles in such terminals. A small number of calcitonin gene-related peptide immunoreactive, presumably primary afferent terminals contained enkephalin-, neurotensin- (and galanin-)like immunoreactivities. These results indicated that galanin can be co-stored with several other neuropeptides in large dense-core vesicles in primary afferent terminals and may presumably be released together with them in the superficial layer of the dorsal horn. Since various combinations of peptides, presumably at varying concentrations, occur in the large dense-core vesicles in a given nerve ending, it is likely that the individual large dense-core vesicles produced in a neuron are heterogenous with regard to peptide content and thus to the message that they transmit upon release.