Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits

DSerotonin 5-HT3 receptors are abundant in the superficial dorsal horn and are likely to have an involvement in processing of nociceptive information. It has been shown previously that 5-HT3 receptors are present on primary afferent terminals and some dorsal horn cells. The primary aim of the present study was to determine what classes of primary afferent possess 5-HT(3)A receptor subunits. We performed a series of double- and triple-labelling immunofluorescence experiments. Subunits were labelled with an anti-peptide antibody and primary afferent axons were identified by the presence of calcitonin gene-related peptide (CGRP) and binding of the lectin IB4. Quantitative confocal microscopic analysis revealed that approximately 10% of axons displaying 5-HT(3)A immunoreactivity were also labelled for CGRP but that only 3% of these fibres bind IB4. We also investigated the relationship between immunoreactivity for the subunit and descending serotoninergic systems, axons originating from inhibitory neurons that contain glutamic acid decarboxylase, and axons of a subpopulation of excitatory neurons that contain neurotensin. None of these types of axon was associated with immunoreactivity for receptor subunits. Ultrastructural studies confirmed that punctate immunoreactive structures observed with the light microscope were axon terminals. These terminals invariably formed asymmetric synaptic junctions with dendritic profiles and often contained a mixture of granular and agranular vesicles. Some terminals formed glomerular-like arrangements. Immunoreactive cells were also examined and were found to contain intense patches of reaction product within the cytoplasm. We conclude that the majority (about 87%) of dorsal horn axons that are immunoreactive for 5-HT(3)A receptor subunits do not originate from the subtypes of primary afferent fibres that bind 1134 or contain CGRP. It is likely that most of these axons have an excitatory action and they may originate from dorsal horn interneurons and/or fine myelinated primary afferent fibres.