The NMDA-NR1 receptor subunit and the mu-opioid receptor are expressed in somatodendritic compartments of central nucleus of the amygdala neurons projecting to the bed nucleus of the stria terminalis

Abstract

The pathway between the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) is emerging as a critical mediator of stress-related affective processes. Evidence also indicates that exposure to drugs of abuse, like opioids, is associated with NMDA-type glutamate receptor-dependent plasticity in the CeA and BNST. However, there is little evidence that NMDA receptors are expressed in CeA neurons projecting to the BNST, or are required for opioid-induced BNST neural activation. Immunoelectron microscopy, tract tracing, and conditional gene deletion technology were used to investigate the synaptic organization of the NMDA receptor and the mu-opioid receptor (μOR) in the CeA-BNST pathway. By dual labeling electron microscopy, numerous CeA-BNST projection neurons expressed the NMDA-NR1 receptor subunit (NR1) or μOR. By triple labeling, it was also found that NR1 and μOR were co-expressed in some CeA-BNST projection neurons. Despite being colocalized in somato-dendritic compartments of CeA neurons, NR1 and μOR were rarely expressed in their axonal terminations in the BNST. Deleting the NR1 gene in CeA neurons resulted in a reduction of morphine-induced Fos protein labeling in the ventral BNST. In summary, NR1 and μOR are coexpressed in somatodendritic sites of CeA neurons, including those projecting to the BNST. In addition, expression of the NR1 gene in CeA neurons is required for morphine-induced BNST neural activation. Thus, postsynaptic NMDA receptors and μORs are positioned for the co-modulation of CeA projection neurons to the BNST, which may provide a synaptic substrate for stress-induced emotional processes critically involved in opioid addictive behaviors.

Fig. 1

Injection of FG in the BNST results in retrograde labeling in CeA neurons(A). Light micrograph illustrating a representative example of FG immunolabeling in the BNST after local administration of the tracer. Immunoperoxidase reaction product for FG is present in the dorsal as well as ventral BNST surrounding the anterior commissure. There is little FG labeling in the regions adjacent to the BNST. (B). Retrogradely labeled neurons are present in the CeA. At high magnification (inset), immunoperoxidase reaction product for FG can be seen throughout the cytoplasm of neuronal cell bodies (arrows). Electron microscopic analysis of FG, NR1, and/or μOR labeling was performed in samples taken from the region of the CeA represented by the area bound by the trapezoid. ac: anterior commissure; d: dorsal; lv: lateral ventricle; m: medial; ot: optic tract. Scale Bar: 1 mm.

Fig. 2

CeA neurons retrogradely labeled after FG administration in the BNST express NR1(A). A soma (NR1+FG-s) expressing diffuse immunoperoxidase reaction product for FG also shows discrete labeling for NR1 (arrows). This cell body contains a large irregularly shaped nucleus (nuc), as well as numerous tubulovesicular organelles characteristic of smooth endoplasmic reticula (vo) and mitochondria (m). Large aggregates of immunoperoxidase reaction product for FG (open arrows) are seen in the cytoplasma. Gold-silver enhanced particles for NR1 (thin arrows) are seen near vesicular organelles, including those associated with mitochondria. Instances of aggregated FG immunoreactivity or NR1 labeling are magnified in the insets. (B). A large dendritic profile (NR1 + FG-d) shows dual immunoreactivity for FG and NR1. Immunoreaction product for FG is widely diffused throughout the intracellular compartment. Immunogold-silver enhanced particles are present near vesicular organelles. (C). A FG labeled dendritic profile also shows immunoreactivity for NR1 (NR1 + FG-d). Immunogold particles for NR1 (thin arrows) are present in intracellular sites of the dendritic shaft, beneath the neck and head of a dendritic spine (sp) that receives an asymmetric synapse (arrow head) from an unlabeled axon terminal (ut). Scale bars: 1 µm (A–B), 0.5 µm (C).

Fig. 3

CeA neurons retrogradely labeled after FG administration in the BNST express μOR(A). A somatodendritic profile (μOR + FG-s) is labeled for FG and μOR. A nucleus (nuc) is seen in the cell body, along with multiple mitochondria (m) that also extend into the proximal dendrite. Multiple immunogold-silver particles for NR1 (arrows) are present in intracellular areas of the proximal dendritic process. This profile is also contacted by unlabeled axon terminals (ut).(B). A small dendritic profile (μOR + FG-d) expresses labeling for μOR and FG. A gold–silver particle (arrow) is present near the plasma membrane beneath the post-synaptic density of an asymmetric type synaptic junction (filled arrow head) formed with an unlabeled axon terminal (ut). Scale bars: 1 µm (A), 0.5 µm (B).

Fig. 4

Different gold–silver enhancement times yield distinct particle sizes(A). Schematic outline of the dual gold–silver enhancement procedure. Sections were incubated with one of the pair of gold-conjugated antisera, which was then silver enhanced for 12 min. This was followed by incubation with the second gold-conjugated antisera, followed by a further 5-minute silver enhancement. This resulted in the first secondary antisera being incubated for 17 min, and the second antisera for 5 min, yielding two distinct but partially overlapping particle size distributions. Arrows represent time periods of silver enhancement, X denotes time period without secondary antisera exposure. The bottom of this figure illustrates representations of silver enhanced gold particle sizes for the two incubation times. Particles are not drawn to scale. (B). Mean particle sizes for each antisera at different silver-enhancement times. These values were obtained in single labeling pilot experiments designed to determine optimal enhancement times. Gold–silver particles in the 5 and 17 minute conditions yielded significantly different particle sizes that could be distinguished visually. (C). Distribution of particle sizes obtained in experiments represented in B above. Although there was some overlap in the particle sizes produced by different silver enhancement times, the vast majority fell into two distinct size ranges. Since most of the particles in the short incubation time were below 40 µm² and most of the large particles were above 100 µm², only particles lower or greater than these values were counted. These criteria minimized, but did not eliminate the possibility of selecting the low number of small particles produced in the long incubation time. Moreover, since there were only a minimal number of particles over 100 µm² with the short incubation time, selecting only the largest particles insured that these came from the appropriate treatment.

Fig. 5

CeA-BNST projection neurons express NR1 and/or μOR(A). In tissue processed for triple labeling, some areas of the neuropil are populated by dendritic profiles expressing NR1 and/or μOR, but not FG. Two dendritic profiles (NR1 ± μOR-d) show labeling for NR1 (thin arrows, long enhancement time) and μOR (thick arrows, short enhancement time). (B). Regions of the CeA processed for triple labeling show neuronal profiles singly labeled for NR1 or μOR, but not FG. A dendritic profile (NR1-d) showing labeling for NR1 (thin arrow, long enhancement time) is contacted by an unlabeled axon terminal. A small presynaptic structure (μOR-a) shows a single particle for μOR (thick arrow, short enhancement time). (C). An area of the CeA processed for triple labeling contains single and dual labeled neuronal profiles. A single labeled dendritic profile (μOR-d) containing a gold–silver particle (thick arrow, long enhancement time) and an NR1 and μOR labeled dendritic profile (NR1 ± μOR-d) are present in the neuropil, which also contains a FG labeled dendrite (FG-d). (D). Triple labeled dendritic profile from a CeA neuron (FG ± NR1 ± μOR-d). Large gold particles for NR1 (thin arrows, long enhancement time) are present near the plasma membrane and clustered intracellularly. A small gold particle for μOR (thick arrow, short enhancement time) is present intracellularly. This profile contains a multivesicular body (mvb) and is contacted by unlabeled axon terminals (ut). (E). Triple labeled dendritic profile of a CeA neuron (FG ± NR1 ± μOR-d). A large silver–gold particle for NR1 (thin arrow, long incubation time) is present near the plasma membrane. A small silver–gold particle for μOR (large arrow, short incubation time) is present intracellularly beneath the shaft of the neck of a spiny process (sp) that receives an asymmetric synapse (arrow head). Scale Bars: 0.5 µm.

Fig. 6

Omission of primary antisera in triple labeling experiments(A). A soma from the CeA is co-labeled for NR1 and μOR (NR1 ± μOR-s). This profile was observed in a control experiment where tissue was processed for triple labeling of NR1, μOR, and FG with omission of the FG primary antisera (No-FG 1°). Despite the lack of immunoperoxidase labeling, large and small immunogold-silver particles for NR1 (thin arrows, long incubation time) and μOR (thick arrows, short incubation time) are visually distinguishable (inset). (B). A cell body from the CeA shows co-labeling for FG and μOR (FG ± μOR-s). This profile was observed when sampling from tissue processed for a control experiment, where sections were prepared for triple labeling of NR1, μOR, and FG with omission of the NR1 primary antisera (No-NR 1°). Diffuse immunoperoxidase FG labeling and large particles for μOR (thick arrows, long incubation time) are seen in this soma (inset). Scale Bar: 1 µm.

Fig. 7

Administration of BDA in the CeA results in anterogradely labeled axonal processes and boutons in the BNST(A–B). As shown by light microscopy, administration of BDA into the CeA resulted in anterograde transport in the BNST. Fibers and punctate granules resembling boutons were present throughout the ventral and dorsal BNST. Areas bound by trapezoids represent regions of the dorsal and ventral BNST sampled by electron microscopy. (C). Electron micrograph showing a BDA labeled axon terminal (BDA-t) contacting the cell body of a dBNST neuron (us). Both Golgi (Gc) and rough endoplasmic reticula (rER) are seen near the nucleus (nuc) of this unlabeled soma. (D). An anterogradely labeled axon terminal (BDA-t) contacts a dendritic profile (ud-1). An unlabeled axon terminal (ut) and dendritic profile (ud-2) are also seen in the adjacent neuropil. (E). A BDA labeled axon terminal (BDA-t) is present in the neuropil with unlabeled dendrites (ud) and axon terminals (ut). The BDA labeled terminal forms an apparent symmetric inhibitory-type type synapse (open arrow head) with one of the dendritic profiles (ud-1). Scale Bars: (Top) 1 mm; (Bottom) 0.5 µm.

Fig. 8

Axon terminals of CeA-BNST projection neurons contact neuronal profiles expressing NR1 or μOR(A). A BDA labeled axon terminal (BDA-t) is present in the dBNST neuropil, which also contains unlabeled dendritic (ud), unmyelinated axonal (un-ma), and axon terminal (ut) profiles. In addition, a dendrite expressing NR1 labeling (NR1-d) is present in close proximity to the anterogradely labeled axon terminal. This dendritic profiles expresses immunogold labeling for NR1 (thin arrows) in intracellular and surface sites. (B). An anterogradely labeled axon terminal (BDA-t) forms a symmetric inhibitory-type synapse (open arrow head) with an NR1 containing dendritic profile (NR1-d) in the dBNST. Immunogold particles for NR1 (thin arrows) are present in intracellular sites of this dendritic profile. (C). In the vBNST, small BDA labeled axon terminals are present in the neuropil with unlabeled or μOR labeled neuronal profiles. A μOR labeled dendritic profile (μOR-d) is contacted by axon terminal profiles expressing BDA (BDA-t1) or BDA and μOR (BDA + μOR-t), as well as a profile devoid of labeling (ut). Immunogold labeling for μOR (thick arrows) is found on the plasma membrane directly adjacent to the BDA labeled terminal, or present near intracellular membranous structures. Also present in the neuropil, a small BDA labeled axon terminal (BDA-t2) contacts an unlabeled (ud) dendritic profile. (D). In the vBNST, a BDA labeled axon terminal profile (BDA-t) contacts two distinct μOR labeled dendritic profiles (μOR-d). It forms a non-synaptic apposition with one dendrite (μOR-d1) and also forms a symmetric inhibitory-type synapse (open arrow head) with the other (μOR-d2). Immunogold labeling for μOR (thick arrows) is present in intracellular sites in both dendrites. Scale Bars: 0.5 µm.

Fig. 9

Deletion of NR1 in CeA neurons results in a decrease in acute morphine-induced Fos expression in the vBNST(A–D). Light micrographs illustrating examples of Fos labeling in the vBNST in morphine administered mice. Area bound by dashed ovals indicates Fos labeling in the vBNST ipsilateral (A) and contralateral (B) to the rAAV-GFP-Cre injection in an area ventral to the CeA (i.e. basomedial amygdala control injection). Expression of Fos is shown in the vBNST ipsilateral (C) and contralateral (D) to the rAAV-GFP-Cre injection in the CeA. (E–F). Expression of GFP in the CeA (E) and BMA (F) after rAAV-GFP-Cre injections. (G). There was a significant reduction in the ratio of ipsilateral/contralateral Fos expression in the vBNST of morphine injected animals receiving rAAV-GFP-Cre in the CeA (Cre-CeA) compared to animals receiving this vector in an area ventral to the CeA (Cre Control), or receiving the rAAV-GFP (GFP CeA) virus in the CeA. Differences were not seen in other brain areas. *p < .05.