Retrograde, trans-synaptic and transneuronal transport of fragment C of tetanus toxin by sympathetic preganglionic neurons

Abstract

The atoxic binding fragment of tetanus toxin, Fragment C, was injected into paravertebral ganglion 14, the avian homologue of the mammalian stellate ganglion. Postinjection survival intervals were varied from 2.5 h to 33 days. Experiments performed at the shortest survival time of 2.5 h showed that Fragment C was retrogradely transported by sympathetic preganglionic axons at a rate ⩾ 10mm/h. At survival times ranging from 5 to 15h, Fragment C-positive, retrogradely labeled sympathetic preganglionic neurons were observed within the last cervical spinal segment and throughout the first three thoracic spinal cord segments. Sporadic retrograde labeling of sympathetic preganglionic neurons was evident within the fourth and fifth thoracic spinal cord segments. Fragment C-labeled perikarya and dendrites exhibited both diffuse cytoplasmic immunostaining as well as intracellular, perinuclear accumulations of small, Fragment C-positive granules. Retrogradely labeled preganglionic neurons were found within both autonomic subnuclei within avian thoracic spinal cord: the column of Terni and the nucleus intercalatus spinalis. The distribution and numerical density of retrogradely labeled sympathetic preganglionic neurons indicated further that: (a) both myelinated and unmyelinated preganglionic axons appear to be capable of intra-axonally transporting Fragment C; and (b) it is unlikely that there is differential Fragment C labeling of a morphologically distinct population of sympathetic preganglionic neurons within or across subnuclei. Fragment C is transferred out of sympathetic preganglionic somas and dendrites into the surrounding neuropil at an aggregate rate ⩾ 5 mm/h. Trans-synaptic transport was evident at postinjection survival times as short as 5 h and continued to increase in density within the sympathetic preganglionic neuropil for 24 h. Fragment C-positive terminal labeling persisted for at least 20 days. At survival times ⩾ 1 day, Fragment C-positive puncta and weak intracellular labeling of neurons were evident in areas of the spinal gray outside of the nuclear boundaries of the column of Terni and nucleus intercalatus. The regions showing evidence of trans-synaptic and transneuronal labeling included: (a) a group of small cells dorsal to the column of Terni, (b) lamina V and (c) lamina VII. This expansion of Fragment C-labeled neuronal elements was segmental in organization and co-extensive with the retrograde labeling pattern of sympathetic preganglionic neurons. Spinal interneurons in these regions may provide segmental, monosynaptic input to sympathetic preganglionic neurons. Fragment C leaked into the systemic circulation from the site of injection in paravertebral ganglion 14. As a result, lamina IX motoneurons were retrogradely and trans-synaptically labeled. Lamina IX labeling was restricted to those motoneurons innervating myotomes which had been surgically damaged. Lamina IX labeling was abolished when the ventral rootlets of spinal segment 14 were cut. Fragment C labeling of afferent terminals within laminae I and II of dorsal horn was also observed. Superficial dorsal horn labeling was abolished following dorsal rhizotomy. These data suggest that Fragment C is anterogradely transported by small caliber myelinated and unmyelinated afferents. There was no labeling of sympathetic preganglionic neurons, lamina IX motoneurons or primary afferent terminations in dorsal horn after intravenous injections of Fragment C in unoperated animals. Human subjects who suffer from moderate to severe tetanus are known to exhibit a rare pathological condition characterized as generalized autonomic hyperactivity. To the extent that Fragment C may be an atoxic surrogate for the identification of neuronal elements intoxicated by tetanus toxin, the present data are consistent with the hypothesis that a principal, perhaps critical, site of action of tetanus toxin in the generation of sympathetic nervous system hyperactivity may be within presynaptic elements contacting sympathetic preganglionic neurons.