Kainate receptors are primarily postsynaptic to SP-containing axon terminals in the trigeminal dorsal horn

Abstract

Kainate receptors (KARs) are involved in the modulation and transmission of nociceptive information from peripheral afferents to neurons in the spinal cord and trigeminal dorsal horns. KARs are found at both pre- and postsynaptic sites in the dorsal horn. We hypothesized that KARs and Substance P (SP), a modulatory neuropeptide that is used as a marker of nociceptive afferents, have a complex interactive relationship. To determine the cellular relationship and connectivity between KARs and SP afferents, we used electron microscopic dual immunocytochemical analysis to examine the ultrastructural localization of KAR subunits GluR5, 6 and 7 (GluR5,6,7) in relation to SP within laminae I and II in the rat trigeminal dorsal horn. KARs were distributed both postsynaptically in dendrites and somata (51% of GluR5,6,7 immunoreactive (-ir) profiles) and presynaptically in axons and axon terminals (45%). We also found GluR5,6,7-ir glial profiles (5%). The majority of SP-ir profiles were presynaptic axons and axon terminals. SP-ir dendritic profiles were rare, yet 23% contained GluR5,6,7 immunoreactivity. GluR5,6,7 and SP were also colocalized at presynaptic sites (18% of GluR5,6,7-ir axons and axon terminals contained SP; while 11% of SP-ir axons and axon terminals contained GluR5,6,7). The most common interaction between KARs and SP we observed was GluR5,6,7-ir dendrites contacted by SP-ir axon terminals; 54% of the dendritic targets of SP-ir axon terminals were GluR5,6,7-ir. These results provide anatomical evidence that KARs primarily mediate nociceptive transmission postsynaptic to SP-containing afferents and may also modulate the presynaptic release of SP and glutamate in trigeminal dorsal horn.

Figure 1

By light microscopy, diffuse GluR5,6,7 immunoreactivity and punctate SP immunoreactivity are found in the superficial trigeminal dorsal horn. A. Some punctate GluR5,6,7 immunoreactivity (filled arrows) are present among the diffuse GluR5,6,7 immunoreactivity in laminae I and II (open arrows). B. Punctate SP immunoreactivity (filled arrows) is present throughout the superficial trigeminal dorsal horn. sp5 = trigeminal tract. Scale bar = 200 μm.

Figure 2

By electron microscopy, GluR5,6,7 immunoreactivity was found in dendrites (A, D), axon terminals (B, D), and glia (C). A. Large and small dendritic processes of the same cell (GluR5,6,7-d) contain cytoplasmic (large arrows) and plasmalemmal (arrowheads) immunogold labeling for GluR5,6,7, but a spiny process that receives an asymmetric synapse (curved arrow) from an unlabeled axon terminal (ut) is not labeled. B. A large GluR5,6,7-ir axon terminal (GluR5,6,7-t) contains small, clear vesicles (scv) and forms an asymmetric synapse (curved arrow) with an unlabeled dendrite. Both cytoplasmic (large arrows) and plasmalemmal (arrowheads) GluR5,6,7 immunogold labeling is present. C. An astrocytic glial process with glial filaments (small arrows) contains GluR5,6,7 immunoreactivity (GluR5,6,7-glia; large arrow) and surrounds an unlabeled axon terminal (ut) that forms an asymmetric synapse (curved arrow). D. A GluR5,6,7-ir axon terminal (GluR5,6,7-t) forms an asymmetric synapse (curved arrow) with a GluR5,6,7-ir dendrite (GluR5,6,7-d). Scale bar = 0.5 μm.

Figure 3

By electron microscopy, GluR5,6,7 immunoreactivity was often found in the dendritic targets of SP-ir axon terminals. A. A large dendrite contains immunogold labeling for GluR5,6,7 at cytoplasmic sites only (large filled arrows). This dendrite receives appositions (open arrows) from several axon terminals containing immunoperoxidase labeling for SP (SP-t1, 2 and 3) and one GluR5,6,7-ir axon terminal (GluR5,6,7-t). B. An axon terminal containing SP (SP-t) in a dense core vesicle (dcv) and an unlabeled terminal (ut) contact a GluR5,6,7-ir dendrite (GluR5,6,7-d). Most of the gold particles are associated with the plasma membrane (arrowheads), particularly along the contact sites with the afferent terminals (open arrows). An immunogold labeled axon (GluR5,6,7-a) is seen in the same field. C. A GluR5,6,7-ir dendrite (GluR5,6,7-d) receives an asymmetric synapse (curved arrow) from an SP-ir axon terminal (SP-t). Gold particles in the GluR5,6,7-ir dendrite are present in the cytoplasm (large arrow) and are associated with the plasma membrane (arrowhead). Scale bar = 0.5 μm.

Figure 4

By electron microscopy, GluR5,6,7 immunoreactivity was sometimes found in glia and presynaptic SP-ir axon terminals. A. A glial process contains immunogold labeling for GluR5,6,7 (GluR5,6,7-glia) and is directly apposed to an SP-containing axon terminal (SP-t) that forms an asymmetric synapse (curved arrow) with an unlabeled dendrite (ud). B. An axon terminal contains both SP and GluR5,6,7 immunoreactivity (SP + GluR5,6,7-t) and forms an asymmetric synapse (curved arrow; far right edge) with an unlabeled dendrite (not shown). An axon terminal containing only immunogold labeling (GluR5,6,7-t) is seen in the same field. Scale bars = 0.5 μm.