Amino acid immunocytochemistry of primary afferent terminals in the rat dorsal horn

Abstract

We combined transganglionic tracing methods with postembedding electron microscopic immunocytochemistry to determine whether identified primary afferent fibers terminating in spinal laminae I-IV may use glutamate and aspartate as neurotransmitters. Sciatic injections of wheat-germ agglutinin conjugated to horseradish peroxidase labeled fine afferent fibers with terminals in laminae I-II of the lumbar spinal cord, whereas injections of the B subunit of cholera toxin conjugated to horseradish peroxidase labeled primary afferent terminals in deeper laminae. Many labeled primary afferent terminals in superficial laminae were involved in glomerular synaptic arrangements; others established nonglomerular contacts. Most glomerular arrangements were clearly immunopositive for glutamate, compared with dendrites, astrocytes, or terminals immunopositive for gamma-aminobutyric acid (GABA). The degree of enrichment varied in labeled terminals of different morphological types. Aspartate was enriched, though to a lesser degree than glutamate, in labeled central terminals of glomeruli in superficial laminae. Labeled primary afferent terminals in laminae III-IV were immunopositive for glutamate, though at lower levels than glomerular terminals in superficial laminae. Aspartate was not enriched in these terminals compared with dendrites, glia, and GABA-positive terminals. These results support a neurotransmitter role for glutamate in primary afferents to the dorsal horn. Quantitative differences in the content of glutamate in identified primary afferent terminals may be related to functional differences. Enrichment of aspartate in terminals in superficial but not deep laminae is compatible with a role for this amino acid in sustained, NMDA-mediated phenomena characteristic of activity in fine caliber afferents.