Ultrastructural visualization and neurochemical characterization of spinal projections of primary sensory afferents from the nipple: combined use of transganglionic transport of HRP-WGA and glutamate immunocytochemistry.
We used transganglionic transport of the neuronal tracer horseradish peroxidase coupled to wheat germ agglutinin (HRP-WGA) and post-embedding immunogold staining to determine the spinal projections and neurochemical identity of sensory afferents originating from a discrete cutaneous area. After SC injection of tracer into the nipple of lactating rats and reaction with tetramethylbenzidine stabilized with diaminobenzidene (TMB-DAB) or DAB and cobalt (TMB-DAB-Co), we found labeled terminals in the internal part of the first two layers of the dorsal horn where they formed axodendritic synapses and, at times, central elements of glomeruli, synaptic complexes believed to be involved in the integration of sensory messages. Immunogold staining of ultra-thin sections of tissue reacted with TMB-DAB-Co revealed that many mammary afferents contained glutamate as putative neurotransmitter. This combined approach thus offers the possibility of marking a limited set of primary afferents, after capture of tracer by sensory receptors of restricted peripheral areas, to visualize their projections at the spinal level and to determine their neurochemical nature with electron microscopy.
Rousselot
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P
Laboratoire de Neuroendocrinologie, Morphofonctionnelle INSERM CJF 91.10, University of Bordeaux II, France.
Poulain
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DA
Theodosis
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DT
D000818:Animals; D005260:Female; D017950:Ganglia, Sensory / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D005971:Glutamates / Q000032:analysis* / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D009475:Neurons, Afferent / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D009558:Nipples / Q000294:innervation*; D051381:Rats; D017208:Rats, Wistar; D014909:Wheat Germ Agglutinins / Q000378:metabolism*
D005971:Glutamates; D014909:Wheat Germ Agglutinins; D006735:Horseradish Peroxidase