Ultrastructural immunocytochemical localization of mu-opioid receptors in rat nucleus accumbens: extrasynaptic plasmalemmal distribution and association with Leu5-enkephalin

Abstract

mu-Opioid receptors and their endogenous ligands, including Leu5-enkephalin (LE), are distributed abundantly in the nucleus accumbens (NAC), a region implicated in mechanisms of opiate reinforcement. We used immunoperoxidase and/or immunogold-silver methods to define ultrastructural sites for functions ascribed to mu-opioid receptors and potential sites for activation by LE in the NAC. An antipeptide antibody raised against an 18 amino acid sequence of the cloned mu-opioid receptor (MOR) C terminus showed that MOR-like immunoreactivity (MOR-LI) was localized predominantly to extrasynaptic sites along neuronal plasma membranes. The majority of neuronal profiles containing MOR-LI were dendrites and dendritic spines. The dendritic plasma membranes immunolabeled for MOR were near sites of synaptic input from LE-labeled terminals and other unlabeled terminals forming either inhibitory or excitatory type synapses. Unmyelinated axons and axon terminals were also intensely but less frequently immunoreactive for MOR. Observed sites for potential axonal associations with LE included coexistence of MOR and LE within the same terminal, as well as close appositions between differentially labeled axons. Astrocytic processes rarely contained detectable MOR-LI, but also were sometimes observed in apposition to LE-labeled terminals. We conclude that in the rat NAC, MOR is localized prominently to extrasynaptic neuronal and more rarely to glial plasma membranes that are readily accessible to released LE and possibly other opioid peptides and opiate drugs. The close affiliation of MOR with spines receiving excitatory synapses and dendrites receiving inhibitory synapses provides the first direct morphological evidence that MOR selectively modulates postsynaptic responses to cortical and other afferents.

Fig. 1.

Photomicrographs of MOR (A) and LE (B) show zones (arrows) of the most intense MOR and LE immunoperoxidase labeling in the NAC. Both the receptor and the peptide show a heterogenous distribution of immunoreactivity within this region. ac, Anterior commissure; d, dorsal; m, medial. Scale bar, 50 μm.

Fig. 2.

Electron micrographs showing adsorption controls for MOR and LE in adjacent tissue sections from the NAC. Aand C show tissue sections processed for immunogold and immunoperoxidase detection of the MOR antibody, respectively.B and D show similarly prepared tissue sections that were immunolabeled using the MOR antibody preadsorbed with the MOR peptide. E shows immunoperoxidase labeling for LE;F illustrates the absence of peroxidase reaction product in adjacent tissue sections, where the LE antibody was incubated with the LE peptide. In A, immunogold particles (small arrows) for MOR are associated with the plasma membrane of two dendrites (MRd_1,2) and an emergent spine head (s) in tissue immunolabeled for MOR. B shows one gold particle (small arrow) that was contained within the cytoplasm of a dendrite in tissue preadsorbed with the MOR peptide. In the same field, a dendritic spine that lacks detectable MOR-LI (us) is apposed to an unlabeled terminal (ut).C shows tissue prepared for MOR immunolabeling, where the peroxidase reaction product for MOR is associated with two dendritic spines (MRs_1,2). The most intense labeling is seen at nonsynaptic regions of the plasma membrane (small arrows). MOR-LI is also seen along specific portions of the membrane of a glial process (asterisks) and in association with vesicles of a small axon (MRa). In D, dendritic spines (us_1,2) lack detectable peroxidase reaction product for MOR in tissue sections preadsorbed with the MOR peptide. Although the nonsynaptic region (small arrows) is devoid of detectable MOR-LI, the synaptic contacts (open arrows) between us_1,2 and two unlabeled terminals (ut) appear similarly electron-lucent to those in C. Additionally, a glial process (asterisks) and small axons (ua) are devoid of immunolabeling for MOR. E shows tissue prepared for LE labeling, where the most intense peroxidase reaction product is in association with axon terminals (Et_1,2).Et₁ is apposed to an unlabeled spine (us), whereas Et₂ is apposed to unlabeled dendrites (ud_1,2). Fillustrates a lack of detectable LE immunoreactivity in tissue that has been preadsorbed with the LE peptide. Scale bars, 0.4 μm.

Fig. 3.

Electron micrographs showing the nonsynaptic localization of MOR-LI in small dendrites and spines. The dendrites (MRd) in A and B are labeled using immunoperoxidase and immunogold–silver, respectively, for detection of MOR-LI. The labeling is largely restricted to nonsynaptic sites in contact with unlabeled terminals (ut andut₁, respectively). The unlabeled terminal (ut₁) in B is also presynaptic to a small spine (MRs) that has one gold particle (arrow), which is near the plasma membrane. The synaptic contacts (open arrows) are both symmetric. No gold–silver particles are seen along portions of the dendrite nearut₂. In C and D, immunoperoxidase and immunogold–silver labeling, respectively, for MOR show mainly extrasynaptic localization of MOR-LI (small arrows) along the plasma membrane of dendritic spines. The spines containing MOR-LI are postsynaptic to unlabeled terminals (ut in C; ut₁ inD); however, in C the asymmetric synaptic junction (large arrow) appears immunolabeled only with the peroxidase method. The asymmetric junction (open arrow) inD is not contacted by gold–silver particles. InD, a second unlabeled terminal (ut₂) is apposed to an unlabeled spine (us). Scale bars, 0.25 μm.

Fig. 4.

Bar graphs showing the subcellular distribution of immunogold–silver particles identifying MOR-LI in dendrites (n = 143) and axons or axon terminals (n = 68) in the NAC. This graph illustrates the distribution of immunogold–silver particles for MOR in contact with (shaded bar) versus not in contact with (white bar) the plasma membrane. From 710 dendritic gold particles, 513 contacted the plasma membrane, and 197 were contained within the cytoplasm. From 200 axonal gold–silver particles for MOR, 122 contacted the plasma membrane and 78 were associated with the cytoplasm. MOR-LI was identified from three to five vibratome sections that were collected from four animals.

Fig. 5.

Electron micrographs in A–C show dendrites containing immunogold-labeling for MOR (MRd) that are apposed to terminals containing peroxidase labeling for LE (Et). The dendrite labeled for MOR-LI in A has one gold–silver particle (top, small arrow) oppositeEt, whereas the majority of particles (small arrows) are located along the plasmalemma of the head and neck of the spine (s) emerging from MRd. This spine receives an asymmetric synapse (open arrow) from an unlabeled terminal (ut). Each of the gold particles in the nearby dendrite and spine are also in contact with plasma membranes.B shows immunogold–silver labeling for MOR associated with both the plasma membrane and cytoplasmic organelles (small arrows) within a dendrite (MRd). MRd is apposed to a terminal showing immunoperoxidase label for LE (Et). In this terminal, the peroxidase is intensely localized to a large vesicle (lv). MRd also receives a symmetric synapse (open arrow) from an unlabeled terminal (ut). In C, a longitudinally sectioned dendrite (MRd) shows gold–silver immunolabeling for MOR (small arrows) localized along the plasma membrane, distal to Et. MRd is also postsynaptic to an unlabeled terminal (ut₁) where the synaptic specialization (open arrow) appears asymmetric. The peroxidase-labeled Et can be compared with a second unlabeled terminal (ut₂) in the same field. Scale bars, 0.3 μm.

Fig. 6.

Electron micrographs showing axonal localization of MOR-LI in relation to unlabeled and LE-labeled terminals. In A (immunoperoxidase) and B(immunogold–silver), single-labeling for MOR is seen primarily along the plasma membrane (small arrows) of axon terminals (MRt), apposed to unlabeled axon terminals (ut_1,2 in A or ut inB). Membranes of small clear vesicles (scv) inMRt of A are lightly labeled, whereas inut₂, small clear vesicles lack MOR-immunoreactivity (USCV). In contrast, the plasma membrane (small arrow) and a saccule of smooth endoplasmic reticulum (ser) in a nearby axon are both intensely immunoreactive. InC and D, respectively, gold–silver labeling for MOR is seen in axon terminals (MRt), which either contain peroxidase reaction product for LE (E) or are apposed to an LE-immunoperoxidase labeled terminal (Et). Both axon terminals show MOR gold–silver particles (small arrows) on or near the plasma membrane. The MRt in C is apposed to several small unlabeled axons (ua) and an unlabeled dendrite (ud). In a nearby small unmyelinated axon (MRa), gold–silver MOR-LI is also seen. Scale bars, 0.2 μm.

Fig. 7.

Immunoperoxidase and immunogold–silver localization of MOR-LI in astrocytic processes (asterisks). The electron micrograph in A shows peroxidase product for MOR localized to discrete portions of the astrocytic plasma membrane (arrows) apposed to an unlabeled axon terminal. The immunogold–silver particles in the micrograph of B show MOR-LI (arrows) mainly within the cytoplasm of an astrocytic process that is contacted by an LE-labeled terminal (Et). Scale bars, 0.3 μm.