Ultrastructural immunolabeling shows prominent presynaptic vesicular localization of delta-opioid receptor within both enkephalin- and nonenkephalin-containing axon terminals in the superficial layers of the rat cervical spinal cord.
Opioid peptides, Met5- and Leu5-enkephalin, are known endogenous ligands for the delta-opioid receptor (DOR) associated with opioid analgesia at the spinal level. To determine the cellular sites for DOR-mediated actions, we examined the ultrastructural localization of DOR and Met5-enkephalin (ME) in the spinal cord by combining immunoperoxidase and immunogold-silver labeling for antibodies against DOR and ME, respectively. Antibodies for DOR localization were raised in guinea pig against peptide 34-47 (p34), an amino acid sequence within the extracellular N-terminus of the DOR recently cloned from mouse neuroblastoma glioma (NG-108) cells. Selective immunoperoxidase labeling for DOR was detected by light microscopy in NG-108 cells and in the lamina I and II of the dorsal horn of the spinal cord (C2-C4). Electron microscopy of these spinal laminae revealed that the majority of the punctate varicosities seen by light microscopy were axon terminals. delta-opioid receptor-like immunoreactivity (DOR-LI) in axon terminals was most prominently associated with large dense core vesicles, and sometimes seen along the membranes of small clear vesicles and segments of the plasmalemma. A semiquantitative analysis of dually labeled sections revealed that of the terminals showing DOR-LI, 23/102 (23%) also contained Met5-enkephalin-like immunoreactivity (ME-LI). Conversely, 23/35 (66%) of the terminals showing ME-LI also showed DOR-LI. In addition to the presynaptic localization, selective postsynaptic densities within dendrites were also occasionally (9%) immunolabeled for the opioid receptor. These results provide the first ultrastructural evidence that DOR may serve autoreceptor functions on ME terminals as well as presynaptic modulation of other transmitters in the dorsal horn of the rat spinal cord. Additionally, the vesicular localization of DOR-LI in axon terminals suggests the involvement of these organelles in the transport of the receptors to the plasma membrane.
Cheng
P Y
PY
Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021, USA.
Svingos
A L
AL
Wang
H
H
Clarke
C L
CL
Jenab
S
S
Beczkowska
I W
IW
Inturrisi
C E
CE
Pickel
V M
VM
D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000378:metabolism*; D003712:Dendrites / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000378:metabolism*; D007106:Immune Sera / Q000276:immunology; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D009333:Neck; D009411:Nerve Endings / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism* / Q000648:ultrastructure; D014018:Tissue Distribution
D007106:Immune Sera; D010446:Peptide Fragments; D017465:Receptors, Opioid, delta; D004744:Enkephalin, Methionine