Calcitonin gene-related peptide and chromogranin a: Presence and intra-axonal transport in lumbar motor neurons in the rat, a comparison with synaptic vesicle antigens in immunohistochemical studies

Abstract

The presence and intra-axonal transport of calcitonin gene-related peptide and chromogranin A were investigated in motor neurons belonging to the rat sciatic nerve. Their co-localization with markers of cholinergic organelles (SV2, p38, and synapsin I) was also investigated, using immunofluorescence techniques, including double labelling experiments. It was found that motor perikarya in the lumbar spinal cord contained calcitonin gene-related peptide-like immunoreactivity and chromogranin A-like immunoreactivity, and probably also caligulin-like immunoreactivity, located in the Nissi substance of the cytoplasm. Also, some SV2 (detected by the monoclonal antibody 10H) was present in some motor neuron perikarya, but most often these were devoid of SV2 and p38, as well as of synapsin I-like immunoreactivity. These three antigens were, on the other hand, concentrated in nerve terminals in the entire gray substance of the spinal cord. In the ventral root, after crushing, calcitonin gene-related peptide, chromogranin A, synapsin I, SV2, p38 and caligulin-like immunoreactivity accumulated in thick and medium-sized axons proximal to the crush, while only antisera against SV2 and p38 labelled accumulated material distal to the crush. In the sciatic nerve, the same essential picture was observed as in the ventral root, but here two other nervous components were also present in the normal sciatic nerve, i.e. peripheral branches of the sensory system and axons of the sympathetic system. By various denervation procedures, it was demonstrated that most calcitonin gene-related peptide-like immunoreactivity and almost all chromogranin A-like immunoreactivity, accumulating in thick axons proximally, emanated from the ventral root. Thin and medium-sized axons originated from the sensory and sympathetic systems and contributed to accumulations both proximally and distally to the crush. Synapsin I-like immunoreactive material accumulated only proximal to the crush, while SV2 and p38-like material accumulated bidirectionally in axons of all sizes. In motor endplates of the rat diaphragm and gastrocnemic muscle, no calcitonin gene-related peptide-like material was observed. However, some chromogranin A-like immunoreactivity was present, in addition to large amounts of synapsin I-like, p38-like and SV2-like material, which had a finely granular appearance and was concentrated near the presynaptic membrane of the nerve terminal endfeet, where synaptic vesicles are known to be located. The results indicate that, in the lumbar motor neuron system of the rat, organelles which carry two matrix components (calcitonin gene-related peptide-like material and chromogranin A-like material), two transmembrane components (SV2 and p38) and at least one surface-associated peptide (synapsin I-like immunoreactivity) are transported with rapid anterograde transport distally, towards the motor endplates. Only the transmembrane components were observed to be retrogradely transported and only the transmembrane components (SV2 and p38) and synapsin I (and traces of chromogranin A) were demonstrated to be concentrated in the motor endplate. The physiological significance of these observations remains to be elucidated.