mu-Opioid receptors are localized to extrasynaptic plasma membranes of GABAergic neurons and their targets in the rat nucleus accumbens

Abstract

The activation of mu-opioid receptors in the nucleus accumbens (Acb) produces changes in locomotor and rewarding responses that are believed to involve neurons, including local gamma-aminobutyric acid (GABA)ergic neurons. We combined immunogold-silver detection of an antipeptide antiserum against the cloned mu-opioid receptor (MOR) and immunoperoxidase labeling of an antibody against GABA to determine the cellular basis for the proposed opioid modulation of GABAergic neurons in the rat Acb. MOR-like immunoreactivity (MOR-LI) was localized prominently to plasma membranes of neurons having morphological features of both spiny and aspiny cells, many of which contained GABA. Of 351 examples of profiles that contained MOR-LI and GABA labeling, 65% were dendrites. In these dendrites, MOR-LI was seen mainly along extrasynaptic portions of the plasma membrane apposed to unlabeled terminals and/or glial processes. Dually labeled dendrites often received convergent input from GABAergic terminals and/or from unlabeled terminals forming asymmetric excitatory-type synapses. Of all profiles that contained both MOR and GABA immunoreactivity, 28% were axon terminals. MOR-containing GABAergic terminals and terminals separately labeled for MOR or GABA formed synapses with unlabeled dendrites and also with dendrites containing MOR or GABA. Our results indicate that MOR agonists could modulate the activity of GABA neurons in the Acb via receptors located mainly at extrasynaptic sites on dendritic plasma membranes. MOR ligands also could alter the release of GABA onto target dendrites that contain GABA and/or respond to opiate stimulation.

Fig. 1.

Electron micrographs showing immunogold MOR labeling and immunoperoxidase labeling for GABA localized to the perikaryon of medium aspiny-type (A) and spiny-type (B) somata (MR + GABAp). InA and B, MOR-LI is identified by dense immunogold–silver deposits (small arrows) distributed along the cytoplasmic surface of the plasma membrane and dispersed within the cytoplasm, sometimes near Golgi lamellae (G) and saccules of endoplasmic reticulum (ser).A, The peroxidase reaction product for GABA is intense and appears diffusely distributed throughout the cytoplasm. GABA immunoreactivity in B is relatively light and is distributed sparsely near the perikaryal plasma membrane (arrowheads). Both somata are apposed to unlabeled terminals (ut), whereas the cell body inA is contacted by glial processes (asterisks). The dually labeled cell body inB is contacted by a GABAergic axon terminal (GABAt). nuc, Nucleus. Scale bars, 0.5 μm.

Fig. 2.

Electron micrographs showing dually labeled dendrites and dendritic spines apposed to GABA-immunoreactive and unlabeled terminals. The dually labeled dendrites in A(MR + GABAd) are apposed to GABA-containing terminals (GABAt_1,2). The three dually labeled dendrites in B (MR + GABAd) are apposed to terminals that lack detectable GABA immunoreactivity (ut). C, Shown is a dendritic spine containing immunolabeling for MOR-LI and GABA (MR + GABAs) contacted by two unlabeled terminals (ut), one of which forms a perforated asymmetric synapse. The axo–spinous complex is enveloped by an astrocytic process (asterisks). In the same field, an unlabeled spine (us) is apposed to an unlabeled terminal (ut) that forms an asymmetric synapse. InA–C, MOR immunogold–silver particles (small arrows) are located primarily along extrasynaptic portions of the dendritic plasma membrane. Scale bars, 0.3 μm.

Fig. 3.

Electron micrographs showing MOR immunogold–silver labeling and immunoperoxidase labeling for GABA in the same or apposed axons and axon terminals. A, A dually labeled axon terminal (MR + GABAt) is apposed to (large arrow) a dendrite that contains MOR-LI (MRd). MR + GABAt also forms a symmetric synapse (curved arrow) with an unlabeled dendrite (ud). In the same field, a presynaptic axon is immunoreactive for GABA (GABAa).B, A dually labeled axon terminal (MR + GABAt) forms a symmetric contact (large arrow) with a dendrite labeled for GABA (GABAd), which is apposed to an astrocytic process (asterisks). InA and B, the axonal MOR immunogold–silver deposits (small arrows) are located mainly near synaptic vesicles and mitochondrial membranes, whereas the dendritic MOR-LI (in A) is localized to the plasma membrane. C, Shown is a MOR-labeled axon terminal (MRt) apposed to a GABA-immunoreactive axon (GABAa). Both axons are apposed to a dendrite containing MOR gold–silver particles (MRd). D, Shown is a bundle of unmyelinated axons, some of them are unlabeled (ua), whereas others are exclusively MOR-labeled (MRa) or GABA-labeled (GABAa). Scale bars, 0.2 μm.

Fig. 4.

Electron micrograph showing immunoperoxidase reaction product for GABA in axon terminals that contact a MOR immunogold-labeled dendrite (small arrows). The MOR-labeled longitudinally cut dendrite (MRd) receives input from two GABA-immunoreactive axon terminals (GABAt_1,2) that form symmetric synapses (large arrows). MRd has an emergent spine head or heads that receive an asymmetric synapse from an unlabeled terminal (ut). s, Spine. Scale bar, 0.27 μm.