Morphological and immunohistochemical studies on the organisation of sacral motoneurons

Abstract

The aim of this thesis was to study pre- and postsynaptic elements during postnatal development and aging. Motoneurons from two adjacent sacral motor nuclei in the cat spinal cord were investigated: the motoneurons innervating intrinsic foot-sole muscles (dorsolateral motor nucleus; DLN) and the motoneurons innervating striated perineal muscles (Onufs nucleus, ventrolateral motor nucleus; VLN). Most of the work has focused on dendrites, since they constitute >90% of the motoneuron's receptive domain. The employed techniques have been intracellular tracer labelling, indirect single- and double staining immunofluorescence, and pre- and post-embedding immunocytochemistry.

The establishment of VLN dendrites in a circumscribed rostro-caudally oriented bundle takes place during the first two postnatal months and appears to be closely linked to the occurrence of dendro dendritic contacts. During maturation a dramatic drop in the proportion of the dendritic membrane area involved in this type of contacts was seen. In the DLN, a postnatal remodelling of the dendrites was seen, including a five-fold increase in total dendritic membrane area, changes in branching pattern and a net decrease in number of branches per dendrite. Dendritic growth/remodelling and neo-synaptogenesis were, however, also seen in aged animals (15y), indicating that neurons and their connectivity are continuously 'remodelled'. Several stigmata of aging were present in the VLN and DLN neuropil of older cats, as the increased frequency of dark-degenerating dendrites, and the occurrence of 'aberrant' dendrites with an irregular outline, increased electron density of the dendroplasm and tighter packing of neurotubules. Regarding presynaptic elements, the distribution and synaptic arrangement of different transmitter identified inputs to the VLN and DLN was examined.

In adult animals, the descending bulbospinal serotoninergic pathway to the VLN was of the same size-order as that to the DLN. Experimental lesions (spinal cord transection at the lower thoracic level performed alone or in combination with unilateral dorsal rhizotomy) revealed a dual supraspinal and spinal origin for the substance P input to both nuclei. The contribution of substance P to the innervation of the VLN and DLN was of the same magnitude, although substance P-immunoreactive (IR) fibres of spinal origin were somewhat more frequent in the VLN. In addition, the VLN received also a denser enkephalinergic innervation than the DLN. Thirty-four % of the boutons apposing VLN dendrites were immunoreactive for glutamate, while 49% of all boutons were immunoreactive for GABA and/or glycine.

The proportions of boutons immunoreactive to glutamate and GABA and/or glycine were rather similar in the VLN and DLN. However, the proportion of boutons single labelled for glycine was greater in the DLN than in the VLN. Substance P-IR boutons had a rather even distribution throughout the dendritic trees of the VLN, apposing both thin distal branches and thick proximal dendrites, while TRH and enkephalin were preferentially located in apposition to more proximal dendritic domains. A frequent finding in the VLN was that one axonal bouton could be seen in synaptic contact with 2-3 dendrites. This divergence of the input at the terminal level was commonly seen among substance P-IR, enkephalin-lR and glutamate-lR boutons, as well as among boutons immunoreactive to GABA- and/or glycine.

A similar arrangement was not observed for TRH, indicating that the supraspinal input to the VLN might rather be a point-to point system with each axonal bouton acting on a single postsynaptic element. In the VLN, differences were seen between animals of different ages in the distribution of amino acid inputs, in particular a smaller number of boutons immunoreactive to glutamate was observed in the older animals. Since no such differences were present in the DLN, this may reflect interindividual variations, changes in the endocrine system and/or differences with age. In the spinal cord of aged rats, axonal profiles showing aging-related morphological changes (accumulation of residual bodies, dystrophy and/or degeneration) were often enriched with immunoreactivity to glutathione (GSH). Since GSH is a scavenger for H202, this suggests that oxidative stress may be relevant for axon degeneration during aging. In addition, about sixty-five percent of the GSH enriched axons with aging-related changes contained also glutamate-immunoreactivity. Aging related degeneration could not be verified for axons containing glycine and/or GABA. This implies that excitatory pathways may be more susceptible to aging-related changes than inhibitory pathways.