Mu opioid receptor localization in the basolateral amygdala: An ultrastructural analysis

Abstract

Receptor binding studies have shown that the density of mu opioid receptors (MORs) in the basolateral amygdala is among the highest in the brain. Activation of these receptors in the basolateral amygdala is critical for stress-induced analgesia, memory consolidation of aversive events, and stress adaptation. Despite the importance of MORs in these stress-related functions, little is known about the neural circuits that are modulated by amygdalar MORs. In the present investigation light and electron microscopy combined with immunohistochemistry was used to study the expression of MORs in the anterior basolateral nucleus (BLa). At the light microscopic level, light to moderate MOR-immunoreactivity (MOR-ir) was observed in a small number of cell bodies of nonpyramidal interneurons and in a small number of processes and puncta in the neuropil. At the electron microscopic level most MOR-ir was observed in dendritic shafts, dendritic spines, and axon terminals. MOR-ir was also observed in the Golgi apparatus of the cell bodies of pyramidal neurons (PNs) and interneurons. Some of the MOR-positive (MOR+) dendrites were spiny, suggesting that they belonged to PNs, while others received multiple asymmetrical synapses typical of interneurons. The great majority of MOR+ axon terminals (80%) that formed synapses made asymmetrical (excitatory) synapses; their main targets were spines, including some that were MOR+. The main targets of symmetrical (inhibitory and/or neuromodulatory) synapses were dendritic shafts, many of which were MOR+, but some of these terminals formed synapses with somata or spines. All of our observations were consistent with the few electrophysiological studies which have been performed on MOR activation in the basolateral amygdala. Collectively, these findings suggest that MORs may be important for filtering out weak excitatory inputs to PNs, allowing only strong inputs or synchronous inputs to influence pyramidal neuronal firing.

Keywords: basolateral amygdala; electron microscopy; immunohistochemistry; interneurons; mu opioid receptor; pyramidal neurons.

Fig. 1

Light micrographs of MOR-ir in the amygdala. (A) Low power photomicrograph of a section through the BLa. Note light MOR-ir in the BLa, intense MOR-ir in the surrounding intercalated nuclei (two of which are indicated by asterisks), and moderate MOR-ir in the central nucleus (Ce). Additional abbreviations: La, lateral nucleus; CP, caudatoputamen. (B) Higher power photomicrograph of a section through the BLa. There are two presumptive interneurons with moderate MOR-ir in this field (larger arrows). Asterisks indicate pyramidal cell nuclei surrounded by perikarya with MOR-ir that is slightly above background . Smaller arrows indicate some of the puncta in the neuropil that may represent axon terminals. In the upper left quadrant of this field are vertically-oriented MOR+ processes with light MOR-ir that resemble dendrites. Scale bars = 200 µm in A, 10 µm in B.

Fig. 2

Histogram showing the number of each MOR+ structure observed in the quantitative analysis of the BLa. So: Somata; LD: large-caliber dendrites (≥1µm); SD: small-caliber dendrites (<1µm); Sp: spines; T: terminals; Ax: axons; G: glial processes.

Fig. 3

Electron micrographs of MOR+ somata in the BLa. Arrowheads indicate examples of granular MOR-ir. (A) A MOR+ soma (M-So; neuron EM03-1 of Table 1) is contacted by a MOR+ terminal (M–t), forming a symmetrical synapse (arrow). MOR-ir is present near the Golgi apparatus in the soma. This soma only received symmetrical synapses suggesting that it is the cell body of a pyramidal neuron. (B) A MOR+ soma (M-So; neuron EM09-1 of Table 1) receiving asymmetrical synaptic contacts (arrows) from two unlabeled terminals (U–t). The presence of asymmetrical synapses suggests that this is the cell body of an interneuron. Scale bars = 0.5 µm.

Fig. 4

Electron micrographs of MOR+ dendrites. Arrowheads indicate examples of granular MOR-ir. (A) A large-caliber MOR+ dendrite (M-LD) and two nearby small-caliber MOR+ dendrites (M-SD). (B) A control section in which the primary MOR antibody was omitted during immunohistochemical processing. Note total absence of granular label in large and small caliber dendrites (LD and SD), spines (sp), and terminals (t). The cisternae with dark membranes (arrows) are easily distinguished from the granular Vector-VIP labeling of MORs shown in A and C. These cisternae may be endosomes. (C) A longitudinally-sectioned large-caliber MOR+ dendrite receives multiple asymmetrical synapses (arrows) from either MOR+ (M–t) or unlabeled (U–t) terminals. The presence of multiple asymmetrical synapses suggests that this dendrite may belong to an interneuron. Scale bars = 0.5µm.

Fig. 5

Electron micrographs of MOR+ small caliber (<1µm) dendrites. Arrowheads indicate examples of granular MOR-ir. (A) A long MOR+ small-caliber dendrite (M-SD) and nearby MOR+ (M–t) or unlabeled (U–t) axon terminals which both form asymmetrical synapses with a MOR+ spine (M-Sp). There is a large-caliber dendrite (M-LD) nearby. (B) Two small-caliber MOR+ dendrites (M-SD), one of which has an unlabeled spine (U-Sp). (C) A small-caliber MOR+ dendrite (M-SD) with an unlabeled spine (U-Sp). Scale bars = 0.5 µm.

Fig 6

Electron micrographs of MOR+ spines. Arrowheads indicate examples of granular MOR-ir. (A) A MOR+ spine (M-Sp) receives an asymmetrical synaptic contact (arrow) from an unlabeled terminal (U–t). (B) A MOR+ spine receives an asymmetrical synaptic contact from an unlabeled terminal (arrow). Three unlabeled spines (asterisks) also receive asymmetrical synaptic contacts from unlabeled terminals. Scale bars = 0.5µm.

Fig. 7

Electron micrographs of various postsynaptic targets of MOR+ terminals. Arrowheads indicate examples of granular MOR-ir. (A) A MOR+ terminal (M–t) forms a symmetrical synapse (arrow) with an unlabeled soma (U-So; neuron EM03-2 of Table 1)). There are two MOR+ small-caliber dendrites (M-SD) nearby. (B) A MOR+ terminal (M–t) form a symmetrical synapse (arrow) with a MOR+ small-caliber dendrite (M-SD). (C) Two MOR+ terminals (M–t) form asymmetrical synapses (arrows) with an unlabeled small-caliber dendrite (U-SD), which also receives an asymmetrical synapse (arrow) from an unlabeled terminal (U–t). Multiple asymmetrical synapses are indicative of interneuronal dendrites. (D) A MOR+ terminal (M–t) forms an asymmetrical synapse (arrow) with a MOR+ spine (M-Sp). (E) A MOR+ terminal (M–t) forms an asymmetrical synapse (arrow) with an unlabeled spine (U-Sp). Scale bars = 0.5µm.

Fig. 8

(A) Electron micrograph of a MOR+ axon terminal (M–t) with 3 MOR-ir granules forming a “flat” symmetrical synapse (arrow) with a MOR+ soma (M-So; neuron EM03-1 of Table 1); the MOR-ir was only associated with the Golgi apparatus in this soma, and is not seen in the field shown. To the right of the axon terminal is a glial process with several granules of reaction product. (B) Electron micrograph of an unlabeled axon terminal (U–t) forming an “invaginated” contact with a MOR+ soma (M-So; neuron EM05-1 of Table 1). There are contacts (arrows) on both sides of the invagination that appear to be obliquely-sectioned symmetrical synapses, identified as such due to their association with clusters of synaptic vesicles directly beneath the contacts, and the presence of densities that appear to be of the intracleft type. Scale bar = 0.5µm for both A and B.

Fig. 9

Histogram showing the number of symmetrical (black) or asymmetrical (dark gray) synapses of MOR+ terminals with various MOR+ (+) or MOR-negative (−) structures. So: somata; LD: large-caliber dendrites (≥1µm); SD: small-caliber dendrites (<1µm); Sp: spines. See Table 2 for exact numerical counts.