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. Author manuscript; available in PMC: 2010 Mar 17.
Published in final edited form as: Neuroscience. 2009 Jan 8;159(2):526–539. doi: 10.1016/j.neuroscience.2008.12.061

Mu-opioid and corticotropin-releasing-factor receptors show largely postsynaptic co-expression, and separate presynaptic distributions, in the mouse central amygdala and bed nucleus of the stria terminalis

Azra Jaferi 1,*, Virginia M Pickel 1
PMCID: PMC2678868  NIHMSID: NIHMS88147  PMID: 19166913

Abstract

The anxiolytic effects of opiates active at the mu-opioid receptor (μ-OR) may be ascribed, in part, to suppression of neurons that are responsive to the stress-associated peptide, corticotropin releasing factor (CRF), in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST). The CRF receptor (CRFr) and μ-OR are expressed in both the CeA and BNST, but their subcellular relationship to each other is not known in either region. To address this question, we used dual electron microscopic immunolabeling of μ-OR and CRFr in the mouse lateral CeA and anterolateral BNST. Immunolabeling for each receptor was detected in the same as well as in separate somatic, dendritic and axonal profiles of neurons in each region. CRFr had a plasmalemmal or cytoplasmic distribution in many dendrites, including those co-expressing μ-OR. The co-expression of CRFr and μ-OR also was seen near excitatory-type synapses on dendritic spines. In both the CeA and BNST, over 50% of the CRFr-labeled dendritic profiles (dendrites and spines) contained immunoreactivity for the μ-OR. However, less than 25% of the dendritic profiles containing the μ-OR were labeled for CRFr in either region, suggesting that opiate activation of the μ-OR affects many neurons in addition to those responsive to CRF. The dendritic profiles containing CRFr and/or μ-OR received asymmetric, excitatory-type synapses from unlabeled or CRFr-labeled axon terminals. In contrast, the μ-OR was identified in terminals forming symmetric, inhibitory-type synapses. Thus, in both the CeA and BNST, μ-OR and CRFr have strategic locations for mediation of CRF and opioid effects on the postsynaptic excitability of single neurons, and on the respective presynaptic release of excitatory and inhibitory neurotransmitters. The commonalities in the synaptic location of both receptors in the CeA and BNST suggest that this is a fundamental cellular association of relevance to both drug addiction and stress-induced disorders.

Keywords: stress, addiction, relapse, withdrawal, anxiety, electron microscopic immunolabeling


Endogenous opioids strongly influence neuroendocrine, autonomic and behavioral responses to stress in a variety of species (Morris et al, 1990; McCubbin et al, 1993; McCubbin, 1993; Sufka et al, 1994; Janssens et al, 1995; Drolet et al, 2001). These effects are primarily mediated by the inhibitory, G-protein-coupled mu-opioid receptor (μ-OR) (Buckingham & Cooper, 1984; Marson et al, 1989; degli Uberti et al, 1995; Liberzon et al, 2002; Xu et al, 2004; Ribeiro et al, 2005). The μ-OR is activated not only by endogenous opioids, but also by the opiates, morphine and heroin (Contet et al, 2004). Relapse to opiate dependence can be induced by stress or by opiate-priming in rodents, and this relapse is attenuated by blockade of the largely stimulatory, G-protein-coupled corticotropin releasing factor receptor (CRFr) (Shaham et al, 1997, Shaham et al, 1998; Lu et al, 2000; Wang et al, 2006). Increased activity in CRF systems within the brain is also thought to underlie the negative affective states and somatic/autonomic symptoms associated with morphine withdrawal (Lu et al, 2000; Iredale et al, 2000; Contarino & Papaleo, 2005; Stinus et al, 2005; Skelton et al, 2007). Together, these observations suggest that opiate withdrawal symptoms may, in part, reflect upregulation of the brain CRF system chronically suppressed by opiates.

The reciprocally-connected central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST) are among the primary brain regions implicated in both the withdrawal symptoms and relapse occurring in animal models of opiate addiction (Heinrichs et al, 1995; Watanabe et al, 2002; Veinante et al, 2003; Nakagawa et al, 2005; Wang et al, 2006; Harris & Aston-Jones, 2007). The emotional responses mediated through the BNST are thought to be slower-onset and longer-lasting, while those involving the CeA are suggested to be rapid-onset and short-duration (Walker et al, 2003; Walker & Davis, 2008). However, the extensive bidirectional anatomical connections between these regions indicate that neither region is independent of the other (Sun et al, 1991; Poulin et al, 2006).

There are many GABAergic and peptidergic neurons throughout the CeA and BNST (Sun & Cassell, 1993; Day et al, 1999). Neurons in each region contain an abundance of opioid peptides and opioid receptors (Moskowitz & Goodman, 1984; Arluison et al, 1994; Mansour et al, 1994; Mansour et al, 1995; Ding et al, 1996; Moriwaki et al, 1996; Day et al, 1999; Abbadie et al, 2000; Chieng et al, 2006; Poulin et al, 2006; Marchant et al, 2007). Opioid receptors, and specifically the μ-OR, show major medial and lateral differences in expression levels (Mansour et al, 1994; Mansour et al, 1995; Abbadie et al, 2000; Poulin et al, 2006). CRF and CRF receptors also are distributed throughout the CeA and BNST (Swanson et al, 1983; Sakanaka et al, 1986; Phelix & Paull, 1990; Uryu et al, 1992; Gray, 1993; Chen et al, 2000; Treweek et al, 2009; Van Pett et al, 2000). CRF is similar to opioid peptides in being differentially expressed in medial and lateral subdivisions (Swanson et al, 1983; Sakanaka et al, 1986; Phelix & Paull, 1990; Uryu et al, 1992; Treweek et al, 2009).

The lateral subdivision of the CeA (CeL) and the anterolateral subdivision of the BNST (BSTal) are homologous regions that are highly interconnected (Sakanaka et al, 1986; Han & Ju, 1990; Sun et al, 1991; Roder & Ciriello, 1993; Sun & Cassell, 1993; Veinante & Freund-Mercier, 1998; Erb et al, 2001; Nakagawa et al, 2005; Poulin et al, 2006). Each of these major subdivisions project extensively to the reward-associated ventral tegmental area (Georges & Aston-Jones, 2002; Dong & Swanson, 2004; Rodaros et al, 2007), as well as to autonomic-related brainstem regions (Moga & Gray, 1985; Moga et al, 1989; Gray & Magnuson, 1987; Gray & Magnuson, 1992; Dong et al, 2001; Dong & Swanson, 2004). The output from the lateral as well as medial subdivisions of both regions is also controlled largely by glutamatergic inputs from the cerebral cortex and basolateral amygdala (Yasui et al, 1991;Schmued, 1994; Walker et al, 2003; Zhu & Pan, 2004; Li and Kirouac, 2008). Interestingly, the CeL and BSTal are among a select group of regions that receive dense, likely glutamatergic, afferent input from the posterior paraventricular thalamus (Frassoni et al, 1997; Li & Kirouac, 2008), a critical regulator of neuroendocrine responses to repeated stress (Jaferi & Bhatnagar, 2006).

Individual activation of the CRFr or μ-OR can modulate excitatory transmission (Liu et al, 2004; Rainnie et al, 2004; Zhu & Pan, 2004; Zhu & Pan, 2005; Orozco-Cabal et al, 2008). It remains unclear, however, whether CRF and opiates modulate the postsynaptic excitability, or presynaptic neurotransmitter release onto single neurons in the CeA or BNST. We examined the dual electron microscopic immunolabeling of the μ-OR and CRFr in single sections through the CeL and BSTal of mouse brain to determine whether these receptors are present in the same or in separate, synaptically-linked neurons in each of these regions. This species was chosen due to the increasing use of mice with genetic manipulations that are of direct relevance to opiate addiction and stress (Kieffer & Gavériaux-Ruff, 2002; Keck et al, 2005). Our results provide the first ultrastructural evidence that the μ-OR and CRFr have subcellular distributions that would enable their participation in the respective opioid and CRF-induced modulation of the postsynaptic excitability of single dendrites and dendritic spines in both the CeL and BSTal. They also suggest that, in each of these regions, there are major differences between the μ-OR and CRFr in their respective involvement in presynaptic release of excitatory compared with inhibitory neurotransmitters. The results have important implications for stress-induced relapse in opiate addicts and for anxiety-related opiate withdrawal symptoms.

Experimental Procedures

Animals and tissue preparation

All experiments were performed in accordance with NIH regulations of animal care, and were approved by the Institutional Animal Care and Use Committee of Weill Medical College of Cornell University. Six adult male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were individually housed and left undisturbed in a quiet facility on a 12-hour light/dark cycle. These mice were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.), and their brain tissue fixed by vascular perfusion through the left ventricle of the heart with sequential delivery of (1) 5 ml of heparin-saline (1000 U/ml), (2) 30 ml of 3.75% acrolein and 2% paraformaldehyde in 0.1M phosphate buffer (PB; pH 7.4), and (3) 100 ml of 2% paraformaldehyde in PB. Brains were removed from the cranium, post-fixed with 2% PFA in 0.1M PB for 30min, and then transferred to PB. These brains were then cut coronally into 40 um sections on a Vibratome (Leica, Deerfield, IL). The coronal sections were collected through the rostrocaudal extent of the CeA and BNST based on the mouse brain atlas by Hof et al (2000).

Antisera

For immunolabeling of the CRFr, we used a rabbit polyclonal antiserum raised against the epitope corresponding to amino acids 230–444 mapping at the C-terminus of the CRFr-1 of human origin (CRF-R1/R2 (H-215): sc-5543 Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The specificity of this antiserum for the CRFr has previously been demonstrated by the diminished immunoreactivity in brain tissue processed after prior adsorption with the antigenic peptide (Treweek et al, 2009), and in brain sections of CRFr-1 (−/−) knockout compared with CRFr-1 (+/+) wildtype mice (Reyes et al, 2008; Treweek et al, 2009).

For immunolabeling of the μ-OR, we used a polyclonal guinea pig antiserum directed against a synthetic peptide corresponding to amino acids 384–398 of the C-terminus of the cloned rat μ-OR (AB1774; Chemicon, Temecula, CA). The specificity of this antiserum has previously been shown by the absence of immunoreactivity in adsorption controls (Rodriguez et al, 2001; Drake & Milner, 2002; Chemicon, 2005). In the present study, we examined immunolabeling for the guinea pig antiserum, as well as for a rabbit μ-OR antiserum (24216; Immunostar Inc, Hudson, WI), in brain sections from μ-OR (+/+) wildtype compared to μ-OR (−/−) knockout mice, each with deletions at distinct exons of the μ-OR gene. These knockouts had deletions at exon 1, exons 2/3 or exon 11, each of which were generously donated by Drs. John Pintar, Sabita Roy and Gavril Pasternak, respectively.

Immunocytochemical labeling

Brain sections were processed for dual immunoperoxidase and immunogold-silver labeling of the CRFr and μ-OR (Chan et al, 1990; Wang et al, 1999; Drake & Milner, 2002; Pickel et al, 2004; Hara & Pickel, 2005; Lane et al, 2008). For this dual-labeling, the vibratome sections were placed in 1% sodium borohydride in 0.1 M PB for 30 minutes to remove excess aldehydes, and then washed thoroughly in 0.1 M PB wash. To enhance the penetration of tissue by immunoreagents, the sections were soaked in a cryoprotectant solution (25% sucrose and 3.5% glycerol in 0.05M PB) for 15 minutes, and rapidly freeze-thawed by sequential immersion in liquid chlorodifluoromethane (Freon, Refron Inc., NY), liquid nitrogen, and 0.1 M PB at room temperature. The sections were then rinsed in 0.1 M Tris-buffered saline (TBS). To reduce non-specific binding of the antisera, the sections were incubated in 0.5% bovine serum albumin (BSA) in 0.1 M TBS, pH 7.6, for 30 minutes before placement in the primary antisera solution. The sections were incubated for 48 hours at 4 C in a solution containing both primary antisera (1:100 rabbit anti-CRFr for immunogold-silver labeling and 1:800 guinea pig anti-μ-OR for immunoperoxidase labeling.) For the reverse labeling scheme, the primary antisera solution consisted of 1:500 rabbit anti-CRFr for immunoperoxidase labeling and 1:500 guinea pig anti-μ-OR for immunogold-silver labeling. All antisera dilutions were prepared in 0.1% BSA in 0.1 M TBS. Incubations with primary as well as secondary antisera were with shaking on a rotator (OS-500; VWR, West Chester, PA).

For immunoperoxidase labeling, the sections were rinsed in 0.1 M TBS and then placed for 30 min in a 1:400 dilution of biotinylated anti-guinea pig (for μ-OR) or biotinylated anti-rabbit (for CRFr). These were then washed in 0.1 M TBS and incubated for 30 min in avidin-biotin peroxidase complex (Vectastain Elite Kit, Vector Labs, Burlingame, CA). This incubation was followed by a wash in 0.1 M TBS, and reaction for 6 min in 0.022% 3,3′-diaminobenzidine (DAB, Aldrich, Milwaukee, WI) and 0.003% hydrogen peroxide in 0.1 M TBS.

For dual detection of antigens, the sections were rinsed in 0.01M PBS (pH 7.4) after the DAB reaction, and blocked in 0.8% BSA and 0.1% gelatin in PBS for 10 minutes. The sections were subsequently incubated with secondary immunogold conjugates for 2 hours at room temperature. These secondary conjugates were a 1:50 dilution of anti-rabbit (for CRFr) or anti-guinea pig (for μ-OR) IgG conjugated with one nm colloidal gold (Amersham, Arlington Heights, IL). The sections were washed several times with 0.1 M TBS. To aid in the binding of gold particles conjugated to IgG, the sections were incubated in 2% glutaraldehyde for 10 minutes. Silver intensification was performed using the IntenS-EM kit (Amersham) with a 6 minute reaction in the silver solution at room temperature. The sections were post-fixed in 2% osmium tetroxide in 0.1 M PB for 60 minutes, washed in PB, and dehydrated by passing through an increasingly concentrated series of ethanol solutions, followed by 100% propylene oxide. The sections were incubated overnight in a 1:1 mixture of propylene oxide and Epon (Electron Microscopy Sciences, Fort Washington, PA), and then transferred to 100% Epon for 2–3 hours before flat-embedding between sheets of Aclar plastic (Electron Microscopy Sciences, Fort Washington, PA). These flat-embedded sections were viewed with a light microscope to select immunolabeled portions of the CeL at Bregma −1.6 mm, and of the dorsal BSTal at Bregma 0.2 mm for collection of ultra-thin sections according to the mouse brain atlas by Hof et al, 2000(Fig. 1). Ultra-thin sections of approximately 70nm thickness were cut from the superficial surface of the section using a diamond knife (Diatome, Fort Washington, PA) on an ultratome (NOVA LKV-Productor AB, Bromma, Sweden). The thin sections were collected on 400-mesh copper grids (Electron Microscopy Sciences), and counterstained with uranyl acetate and lead citrate (Reynolds, 1963). These grids were rinsed and allowed to air dry before viewing at 60 kV with a Philips CM10 transmission electron microscope (FEI, Hillsboro, OR). Those areas showing immunolabeling of μ-OR and CRFr were then magnified and captured as digital images using AMT Advantage HR/HR-B CCD Camera System (Advanced Microscopy Techniques, Danvers, MA).

Fig. 1.

Fig. 1

The BNST and CeA subregions sampled for electron microscopy are shown in plastic-embedded vibratome hemisections of a mouse brain at the level 0.2 mm anterior to Bregma for the BNST and 1.6mm posterior to Bregma for the CeA (Hof et al, 2000). The trapezoids indicate the locations from which the ultrathin sections were collected for electron microscopic image analysis. Scale bar = 100 μm. Lv= lateral ventricle, BSTal= anterolateral bed nucleus of the stria terminalis, ac= anterior commisure, cp= caudate putamen, BLA= basolateral amygdala, CeL= lateral subdivision of the central amygdala, CeM= medial subdivision of the central amygdala, ot= optic tract.

Electron Microscopic Analysis

Electron microscopic analysis was carried out in ultra-thin sections of six vibratome sections (three each of CeA and BNST from three mice). To confirm that the μ-OR and CRFr have similar labeling patterns regardless of the immuno-markers used, we repeated this analysis using the reverse labeling scheme in an additional six vibratome sections from three mice. Micrographs containing μ-OR and CRFr immunoreactivity at magnifications of 19000X to 25000X were analyzed in a total tissue area of 4889 μm2 in the CeL and 4881 μm2 in the BSTal. We exclusively examined tissue at the surface of vibratome sections at the Epon-tissue interface, where there was optimal penetration of primary and secondary antisera. All electron microscope images were imported into an IBM computer, where Adobe Photoshop (version 7.0.1, Adobe Systems Inc., Mountain View, CA) was used to sharpen and enhance contrast as needed. These images were then imported into PowerPoint (Microsoft Office, 2007), which was used to assemble figures and add lettering.

Profiles containing μ-OR and/or CRFr immunoreactivity were classified as neuronal (dendrites, dendritic spines, axons, axon terminals) or glial processes based on criteria described in Peters et al (1991). Immunoperoxidase labeling was regarded as positive when the electron-dense reaction product in individual profiles was greater than that seen in other morphologically similar profiles in the neuropil. Profiles were considered to be immunogold-labeled when they contained one or more gold particles. Profiles considered immunogold-labeled when containing only one gold particle included small (< 0.6 μm) structures, and were always located in a neuropil in which no presumed extraneous particles were seen in a comparable area of neuropil. This approach was validated by examining spurious gold particles overlying myelin, a structure not known to express receptors. Out of a total of 122 myelinated axons in 2,448 μm2 in the CeL and 2,428 μm2 in the BSTal examined, none displayed CRFr immunogold particles over myelin. In each subregion, we quantified the number of neuronal processes that were single- or dual-labeled as well as the number and types of synaptic associations between profiles that were separately labeled for each receptor. Asymmetric synapses were recognized by thick postsynaptic densities, while symmetric synapses were recognized by equally thin pre- and postsynaptic specializations. Appositions/non-synaptic contacts were recognized by closely spaced, parallel plasma membranes that lacked discernible synaptic densities.

Results

Regional and cellular location of μ-OR

The light microscopic immunoperoxidase labeling of the μ-OR was prevalent in perikarya and processes of the CeL as well as medial and capsular subdivisions of the CeA, and in the anterolateral subdivision of the BNST. In each region, the μ-OR immunoreactivity was detected in fewer perikarya and processes in knockout mice with deletions of Exon 1 (Fig. 2B, 2C), Exons 2/3 or Exon 11 (not shown). The residual labeling suggests that the guinea pig μ-OR antiserum recognizes these and possibly other exons within the μ-OR (Abbadie et al, 2000). Like the guinea pig antiserum, the commonly used rabbit μ-OR antiserum also produced residual immunoperoxidase labeling of perikarya and processes in the CeA and BNST of Exon 1, 2/3 and 11 knockout mice. However, with the rabbit antiserum, immunolabeling in these knockout mice was equivalent to, if not more than, labeling seen in μ-OR wildtype mice, suggesting cross reactivity of the antiserum with proteins other than the mu-opioid receptor. The CRFr was less abundant, but otherwise similar to the μ-OR in being expressed in both perikarya and processes in the CeL and BSTal.

Fig. 2.

Fig. 2

Light micrographs showing immunoperoxidase labeling of the μ-OR in the CeL (A, B) and BSTal (C, D). In an exon-1 wildtype (+/+) mouse, dense immunoperoxidase labeling of the μ-OR is seen in perikarya (P) as well as in thick, smooth dendritic-like structures and in punctate processes (arrow) resembling varicosities (A, C). In an exon-1 (−/−) mouse, the μ-OR labeling is seen in fewer perikarya and processes in each of the respective regions (B, D). BSTal= anterolateral BNST; CeL= lateral subdivision of central amygdala. Scale bars = 50 μm.

Electron microscopy showed the presence of the μ-OR and CRFr in the same as well as separate somatic, dendritic and axonal profiles in the CeL and BSTal. In contrast with the prominent neuronal localization, less than 4% of the μ-OR or CRFr was seen in glial profiles of either region.

Somatodendritic distributions of μ-OR and CRFr

The pattern of immunolabeling for the μ-OR and/or CRFr in somatic and dendritic profiles was similar in the CeL and BSTal. Thus, the described subcellular distributions are equally applicable to both regions except when specifically referred to in the following descriptions. The μ-OR was prominent on non-synaptic portions of plasma membrane (Fig. 3A), and within cytoplasm (Fig. 3B), as shown by immunogold or peroxidase labeling (Fig. 3C). Immunoreactivity for the μ-OR (Fig. 3C, 3D) or CRFr (Fig. 4, 5A) was observed in dendrites and dendritic spines. Seventy-seven percent (270/350) of all μ-OR-labeled neuronal (dendritic + axonal) processes observed in the CeA were dendrites and dendritic spines. Similarly, in the BSTal, 79% (309/389) of the total neuronal processes containing the μ-OR were dendrites and spines.

Fig. 3.

Fig. 3

Electron micrographs showing immunogold or immunoperoxidase labeling of the μ-OR in neuronal and glial profiles without detectable CRFr in the mouse CeL (A, B, D) and BSTal (C). μ-OR immunogold particles (filled-head arrow) are distributed in dendrites (μOR-De) along the plasma membrane (A) or in the cytoplasm (B). In (B), μ-OR immunogold labeling is observed on the plasma membrane in an axon terminal (μOR-Te) that contains a few small, clear vesicles, and many large, dense core vesicles (white block arrow). In (C), μ-OR immunoperoxidase labeling is diffusely distributed along the plasma membrane, and in the cytoplasm of a dendrite and dendritic spine (μOR-Sp). Plasmalemmal CRFr immunogold labeling (open-head arrow) in (C) is seen in an axon terminal (CRFr-Te) that contains densely packed small, clear vesicles, and forms an asymmetric synapse with a dendritic spine that contains the μ-OR (μOR-Sp). Asymmetric, excitatory-type (arrowhead) and/or symmetric, inhibitory-type (curved arrow) synapses are formed by unlabeled terminals (ut) presynaptic to μ-OR-labeled dendrites and spines in (B), (C) and (D). The μ-OR immunoperoxidase (C) or immunogold (D) labeling is also seen in glial processes that appose μ-OR-labeled dendrites. Scale bars = 0.5 μm.

Fig. 4.

Fig. 4

Electron micrographs showing immunogold labeling of the CRFr (open-head arrow) and immunoperoxidase labeling of the μ-OR within the same dendrites of the mouse CeL (A, B, C) and BSTal (D). CRFr immunogold particles are located in the cytoplasm (A) and on the plasma membrane (B, C, D) of dendrites that are immunoperoxidase labeled for the μ-OR (Dual-De). In each micrograph, the dual-labeled dendrites receive asymmetric, excitatory-type synapses (arrowhead) from unlabeled axon terminals (ut). Dual-labeled dendrites also appose terminals that contain CRFr immunogold labeling (CRFR-Te) in (C) and (D). In (A), the dual-labeled dendrite receives a symmetric, inhibitory-type synapse (curved arrow) from an unlabeled terminal. Scale bars = 0.5 μm.

Fig 5.

Fig 5

Immunogold localization of the CRFr (open-head arrow) or μ-OR (filled-head arrow) near asymmetric, excitatory-type synapses on dendritic spines of the mouse CeL (A, D) and BSTal (B, C). In (A) and (B), the CRFr immunogold labeling is seen in the region of the asymmetric postsynaptic membrane (arrowhead) on spines that are immunoperoxidase labeled for the μ-OR (Dual-Sp). μ-OR immunogold particles have a similar location near asymmetric synapses in dendritic spines (μOR-Sp) in (C) and (D). Spines containing the μ-OR (C, D) or both receptors (A, B) receive asymmetric synapses from unlabeled axon terminals (ut). In (D), a dendrite that is immunoperoxidase labeled for the μ-OR receives a symmetric, inhibitory-type synapse (curved arrow) from an unlabeled terminal. Scale bars = 0.5 μm.

Like μ-OR-containing profiles, the majority of observed CRFr-containing neuronal profiles were dendrites and dendritic spines [62% (88/141) for CeL; 63% (90/143) for BSTal]. Many dendrites (Fig. 4) and spines (Fig. 5A, 5B, 6C) containing immunogold labeling for the CRFr also displayed immunoperoxidase labeling for the μ-OR. Although the numbers of dendritic profiles containing the CRFr and/or μ-OR varied between individual mice (Table I), the proportion of single- or dual-labeled dendritic profiles were similar between mice as well as on average. Out of all CRFr-labeled dendritic profiles in the CeL, approximately 66% (58/88) contained immunoreactivity for the μ-OR (Table II). Similarly, in the BSTal, approximately 63% (56/90) of all dendritic profiles containing CRFr immunoreactivity also displayed μ-OR labeling. Conversely, when considering the total number of μ-OR-labeled dendritic profiles that contained the CRFr, 21% (58/270) were dual-labeled in the CeL, and 18% (56/309) were dual-labeled in the BSTal (Table II).

Fig. 6.

Fig. 6

Electron micrographs showing the post- and/or presynaptic localization of the CRFr or μ-OR in axon terminals of the mouse CeL (B, C, D) and BSTal (A, E). Unlabeled or CRFr-labeled axon terminals (CRFR-Te) form asymmetric, excitatory-type synapses (arrowhead) with dendritic spines containing the CRFr (A), the μ-OR (B), or both receptors (C). In (B), CRFr immunogold particles (open-head arrow) have cytoplasmic locations in an axon terminal that contains a large, dense core vesicle (white block arrow) near the synapse. In (C), CRFr immunogold particles are seen on the plasma membrane of axon terminals (CRFR-Te), one of which forms a clearly defined asymmetric, excitatory-type synapse on a large spine head that is dual-labeled (Dual-Sp) with CRFr immunogold and μ-OR peroxidase. μ-OR immunoperoxidase labeling is seen in a small axon (μOR-Ax) in (C), and at a presynaptic location in an axon terminal that forms a symmetric synapse with an unlabeled dendrite (ud) in (D). μ-OR immunogold particles (filled-head arrow) are distributed on the plasma membrane of a non-synaptic axon terminal in (E). MA = myelinated axons. Us = unlabeled spine. Scale bars = 0.5 μm.

Table I. Summary of Samples.

Summary of the number of neuronal profiles sampled from each mouse. Sections of the CeL or BSTal from mice 1–3 were processed for immunogold labeling of the CRFr and immunoperoxidase labeling of the μ-OR. Sections from mice 4–6 were processed with the reverse markers (CRFr peroxidase and μ-OR immunogold).

Mouse #1 Mouse #2 Mouse #3
μOR CRFr CRFr+ μOR μOR CRFr CRFr+ μOR μOR CRFr CRFr+ μOR
CeL
# of Dendritic Profiles 146 29 52 262 24 67 230 38 54
# of Axonal Profiles 61 71 4 118 30 0 55 53 2
BSTal
# of Dendritic Profiles 308 30 64 263 41 53 188 29 52
# of Axonal Profiles 76 49 4 84 57 7 63 40 4
Reverse Markers Mouse #4 Mouse #5 Mouse #6
μOR CRFr CRFr+ μOR μOR CRFr CRFr+ μOR μOR CRFr CRFr+ μOR

CeL
# of Dendritic Profiles 122 27 42 106 29 45 172 32 45
# of Axonal Profiles 98 49 0 64 31 1 64 48 1
BSTal
# of Dendritic Profiles 79 46 45 78 27 35 70 40 43
# of Axonal Profiles 19 41 3 27 26 1 23 28 0

Table II. Distribution of CRFr and μ OR within neuronal profiles.

Summary of the mean percentages of axonal (axons and axon terminals) and dendritic (dendrites and spines) profiles that were immunogold labeled for the CRFr, immunoperoxidase labeled for the μ-OR, or labeled for both receptors in sections of the mouse CeL or BSTal.

Dendritic profiles (single label) Dendritic profiles (dual label) Axonal profiles (single label) Axonal profiles (dual label)
CeL
CRF receptor 34% (30/88) 66% (58/88) 96% (51/53) 4% (2/53)
Mu-opioid receptor 79% (213/270) 21% (58/270) 97% (78/80) 3% (2/80))
BSTal
CRF receptor 37% (33/90) 63% (56/90) 91% (49/54) 9% (5/54)
Mu-opioid receptor 82% (253/309) 18% (56/309) 94% (74/79) 6% (5/79)

These patterns of labeling in the CeL and BSTal were confirmed by repeating the immunolabeling procedure with the reverse markers (Fig. 5C, 5D). In both the CeL and BSTal, more than half of all dendritic profiles that were immunoperoxidase-labeled for the CRFr also contained μ-OR immunogold particles [60% (44/73) for CeL; 52% (41/79) for BSTal]. In contrast, a minority of all μ-OR-labeled dendritic profiles contained CRFr immunoreactivity [25% (44/177) for CeL; 35% (41/117)] for BSTal].

Immunogold particles identifying the μ-OR or CRFr were often observed within or near the postsynaptic membrane specializations at asymmetric synapses on dendritic spines in both the CeL and BSTal (Fig. 5). Out of 59 unlabeled terminals in contact with dendrites containing one or both receptors in the CeL, approximately 31% (18/59) formed asymmetric synapses with dual-labeled dendrites, and 10% (6/59) formed symmetric, inhibitory-type synapses with dual-labeled dendrites (Table III). Forty-seven percent of these terminals (28/59) formed asymmetric synapses, and 7% (4/59) formed symmetric synapses with μ-OR-labeled dendritic profiles. Five percent (3/59) formed asymmetric synapses with CRFr-labeled dendritic profiles. The BSTal was remarkably similar to the CeL with respect to types of synaptic associations of dendrites containing one or both receptors. For example, in the BSTal, approximately 30% (19/65) of the unlabeled terminals formed asymmetric synapses, and 8% (5/65) formed symmetric synapses with dual-labeled dendrites (Table III). This suggests that the CRFr and μ-OR are mainly segregated to those dendritic segments that are receptive to excitatory inputs as indicated by the asymmetry of the postsynaptic membranes. It should be noted, however, that in some cases as in Figure 4A, the symmetry/asymmetry of the synapses can be difficult to judge due to the accumulation of peroxidase product at postsynaptic densities.

Table III. Synaptic input to CRFr- and/or μ OR-labeled dendritic profiles.

Summary of the types of synapses (asymmetric, excitatory-type or symmetric, inhibitory-type) that CRFr- and/or μ-OR-containing dendritic profiles received from unlabeled axon terminals. These dendritic profiles were immunogold-labeled for the CRFr, immunoperoxidase-labeled for the μ-OR, or labeled for both receptors in sections of the mouse CeL or BSTal. The number of axon terminals observed in the CeL (n=59) was averaged across three mice where n=43 for mouse 1, n=58 for mouse 2, and n=77 for mouse 3. The number of axon terminals observed in the BSTal (n=65) was also averaged across three mice where n=79 for mouse 1, n=72 for mouse 2, and n=44 for mouse 3.

CRFr- labeled dendritic profiles μOR-labeled dendritic profiles Dual-labeled dendritic profiles
CeL
Asymmetric synapse 5% (3/59) 47% (28/59) 31% (18/59)
Symmetric synapse 0% (0/59) 7% (4/59) 10% (6/59)
BSTal
Asymmetric synapse 11% (7/65) 42% (27/65) 29% (19/65)
Symmetric synapse 2% (1/65) 8% (5/65) 8% (5/65)

Axonal distributions of μ-OR and CRFr

The pattern of immunolabeling for the μ-OR or CRFr in axonal profiles was similar in the CeL and BSTal. These included small axons (Fig. 6C) and axon terminals (6B-6E). The axonal immunogold particles identifying the μ-OR or CRFr were in contact with the plasma membrane (Fig. 6C, 6E), or were distributed over vesicular structures in the cytoplasm of axon terminals (Fig. 6B). μ-OR or CRFr-immunoreactive axon terminals contained mainly small, clear synaptic vesicles that typically store amino acid neurotransmitters (i.e., glutamate, GABA) (Peters & Palay, 1996; Torrealba & Carrasco, 2004). Some of these terminals, however, contained a mixture of small clear and large, dense core vesicles (Fig. 3B, 6B), the latter of which typically store monamines or peptides (i.e., opioids, CRF) (Nirenberg et al, 1995; Peters & Palay, 1996; Torrealba & Carrasco, 2004).

The axonal profiles containing each of these receptors were less numerous than the labeled dendritic profiles described above. In the CeL, small axons or axon terminals comprised approximately 23% (80/350) of all identified μ-OR-labeled neuronal profiles, and 20% (79/389) of those in the BSTal. Out of all CRFr-labeled neuronal processes, 38% (53/141) were axonal profiles in the CeL, and similarly, 38% (54/143) were axonal profiles in the BSTal.

Dual-labeling of the CRFr and μ-OR was rarely observed in axon terminals in either the CeL or BSTal. The number of axonal profiles labeled for the CRFr and/or μ-OR varied between mice (Table I), but the proportion of single- and dual-labeled axonal profiles was similar between mice, and on average. Approximately 4% (2/53) of all CRFr-labeled axonal profiles in the CeL contained immunoreactivity for the μ-OR (Table II). In the BSTal, 9% (5/54) of the axonal profiles that contained the CRFr also showed μ-OR-labeling (Table II). Conversely, 3% (2/80) of all μ-OR-labeled axonal profiles also contained the CRFr in the CeL, and 6% (5/79) contained both receptors in the BSTal. This rarity of dual-labeling of the CRFr and μ-OR in axonal profiles was confirmed by immunolabeling with the reverse markers. The largely separate distribution of the CRFr and μ-OR in axon terminals is consistent with their respective preferential localization in axon terminals that form asymmetric compared with symmetric synapses.

In both the CeL and BSTal, axon terminals that were CRF-immunoreactive or unlabeled often formed asymmetric synapses with dendritic profiles containing exclusively the μ-OR (Fig. 3C, 5D, 6B) or CRFr (Fig. 6A) as well as those containing both receptors (Fig. 4C, 5A, 6C). The asymmetric synapses are typical of axon terminals containing glutamate (Farb et al, 1992). In contrast, the μ-OR-labeled terminals in each region commonly showed non-synaptic appositions or formed symmetric synapses with unlabeled dendrites (Fig. 6D). Numerous non-synaptic appositions were observed between μ-OR and/or CRFr-immunoreactive axon terminals, and dendrites containing both receptors in the CeL and BSTal. In the CeL, 106 non-synaptic appositional contacts were observed between dual-labeled dendrites and axon terminals. Approximately 67% (71/106) of these terminals were unlabeled in the CeL. In the BSTal, approximately 61% (47/77) of the appositional contacts were between dual-labeled dendrites and unlabeled terminals.

Discussion

The present study provides the first ultrastructural evidence that in the mouse CeL and BSTal, the μ-OR and CRFr are co-expressed in dendritic profiles receiving asymmetric, excitatory-type synapses (Fig. 7.1). These results suggest that activation of the μ-OR or CRFr may independently or opposingly affect the postsynaptic excitability of neurons in two brain regions that are critical mediators of the stress response. In addition, the observed presence of the CRFr within some of the excitatory-type terminals that are presynaptic to μ-OR and/or CRFr-containing dendrites in each region suggests that the CRFr can also affect the output of these neurons through presynaptic mechanisms independent of the μ-OR (Fig. 7.2). Instead, the μ-OR is identified in axon terminals forming inhibitory-type synapses with mainly unlabeled dendrites (Fig. 7.3). Collectively, these results have important implications for the well-documented functional associations between stress and opiate abuse.

Fig. 7.

Fig. 7

Schematic diagram showing the distribution of the CRFr and μ-OR in the same postsynaptic, and separate presynaptic, neurons of the CeL and BSTal. Both the μ-OR and CRFr are present within or near excitatory-type synapses (+) on dendritic spines (1), suggesting that the activation of the μ-OR or CRFr can independently or opposingly affect the postsynaptic excitability of single neurons in each of these regions. These excitatory-type synapses are formed by axon terminals that are unlabeled or that contain the CRFr (CRFr-Te). Axon terminals that contain the CRFr also form excitatory-type synapses with μ-OR-expressing dendritic spines (2), suggesting that the CRFr can affect the output of opiate-sensitive spines in the CeL and BSTal through presynaptic mechanisms. The μOR-containing axon terminals (μOR-Te) are illustrated as arising from intrinsic or extrinsic inhibitory neurons whose local dendrites express μ-OR, but not CRFr, and as forming inhibitory-type synapses (−) with dendrites not expressing either receptor (3). In contrast, the CRFr is shown in axon terminals that form excitatory-type synapses, suggesting distinct roles for the μ-OR and CRFr in the release of inhibitory and excitatory neurotransmitters, respectively.

Methodological considerations

The CRFr antiserum has previously (Reyes et al, 2008; Treweek et al, 2009), and the guinea pig μ-OR antiserum has presently, been shown to recognize proteins that are absent or attenuated in the respective CRFr-1 and μ-OR knockout mice. Our present results indicate that the μ-OR antiserum recognizes multiple exons, since no one gene knockout completely eliminated the immunolabeling. However, we cannot totally exclude the possibility that at least some of the residual immunolabeling observed in knockout mice reflects cross-reaction of the antiserum with structurally similar proteins. The potential cross reaction, or recognition of multiple exons at the c-terminus of the cloned μ-OR with the guinea pig antiserum makes it necessary to consider the labeling as μ-OR-like immunoreactivity, a caveat also applicable to the CRFr antiserum which may partially recognize the CRFr-2 (Treweek et al, 2009). Both antisera do, however, meet other important criteria of specificity including absence of immunoreactivity when pre-adsorbed with the antigenic peptide (Rodriguez et al, 2001; Drake & Milner, 2002; Chemicon, 2005; Treweek et al, 2009). The pattern of labeling obtained with the guinea pig μ-OR antiserum is also consistent with μ-OR autoradiographical localization (Mansour et al, 1987), and with the distribution of other μ-OR antisera that have been used extensively in rodent brain (Arvidsson et al, 1995; Mansour et al, 1995; Wang et al, 1996; Wang et al, 1999).

In most cases, only a few (1–4) immunogold particles were seen in neuronal profiles that were considered to be labeled for either receptor. This is a valid approach only in samples of brain tissue in which structures such as myelin show few, if any, spurious gold particles, and when there is a correspondence between the subcellular location seen with immunogold and immunoperoxidase labeling methods (Wang & Pickel, 2001; Wang & Pickel, 2002; Hara & Pickel, 2005). The moderate to low amount of CRFr labeling in the CeA is consistent with known low levels of receptor mRNA in this brain region (Van Pett et al, 2000), which contains an abundance of CRF immunoreactive neurons (Swanson et al, 1983; Uryu et al, 1992; Gray, 1993; Treweek et al, 2009). Low expression levels of the CRFr and/or μ-OR may have contributed to an underestimation of co-localization of these receptors in single neurons, as well as the synaptic associations occurring between neurons that differentially express these receptors.

Co-localization of μ-OR and CRFr in dendrites receiving excitatory-type synapses

In both CeL and BSTal, a similar percentage of CRFr-expressing dendrites contain the μ-OR. In addition, μ-OR and/or CRFr-expressing dendrites in each brain region more often receive excitatory-type than inhibitory-type synapses. These regional similarities in receptor distribution and synaptic associations in the CeL and BSTal are in line with previous findings of a correspondence between the CeA and BNST in cytologic features (Cassell & Gray, 1989; Moga et al, 1989), anatomical connectivity (Gray and Magnunson, 1987; Gray & Magnunson, 1992; de Olmos & Heimer, 1999; McDonald et al, 1999; Cassell et al, 1999; Shammah-Lagnado et al, 2000), and neurochemical attributes (Shimada et al, 1989; Sun & Cassell, 1993; Day et al, 1999), as well as functional roles in emotional and autonomic processes (Saper, 1995; Koob, 2003).

Our present findings provide evidence that the CRFr is prevalent within or near excitatory synapses in dendritic compartments that also contain the μ-OR in the CeL and BSTal. The presence of both the μ-OR and CRFr at these synapses suggests that the activation of one or both receptors can affect the postsynaptic excitability of neurons in each of these regions. The activation of the μ-OR and CRFr may, however, have opposing effects on intracellular signaling. This is consistent with the prior suggestion that endogenous opioids may counterbalance the excitatory effects of CRF on stress systems in order to limit excessive activation of the stress response (Curtis et al, 2001; Valentino & Van Bockstaele, 2001). The functional opposition is supported by distinct intracellular signaling mechanisms. Opioids, specifically enkephalin, act on the Gi/Go-coupled μ-OR to inhibit the cAMP pathway, while CRF preferentially acts on the Gs-coupled CRFr to stimulate the cAMP pathway (Ueda et al, 1988; Grammatopoulos et al, 2001).

Presynaptic distributions of μ-OR and/or CRFr

Co-localization of the μ-OR and CRFr was rarely seen in CeL or BSTal axons or terminals, although numerous axonal profiles showing single labeling were seen in each region. The μ-OR immunoreactivity was evident on plasma membranes of axon terminals that contained dense core vesicles, suggesting a role for the μ-OR in regulation of the release of monoamines or neuropeptides that are frequently stored in these vesicles. The endogenous opioids and CRF are among the many neuropeptides in axon terminals of the CeL and BSTal (Swanson et al, 1983; Uryu et al, 1992; Poulin et al, 2006; Treweek et al, 2009). Presynaptic μ-OR autoreceptor in axon terminals that contain enkephalin-type opioids has been described in other brain regions such as the ventral tegmental area, nucleus accumbens and caudate-putamen as well as the spinal cord dorsal horn (Cheng et al, 1996; Svingos et al, 1996; Wang et al, 1996; Garzon & Pickel, 2002). In addition, however, we also observed the μ-OR in axon terminals that contained small, clear vesicles that are typical of those that release amino acid neurotransmitter (Sesack & Pickel, 1995; Peters & Palay, 1996; Torrealba & Carrasco, 2004), and often forming symmetric synapses. Together, these observations suggest that enkephalin and/or GABA or other inhibitory amino acids are the neurotransmitters in many of the neurons that express the μ-OR in their axon terminals. The CRFr-containing axon terminals formed excitatory-type synapses, a type that rarely contained the μ-OR.

Implications

The level of co-expression of the CRFr and μ-OR in the CeL and BSTal is above that for most other receptors within any brain region thus far examined (Huang & Pickel, 2003; Delle Donne et al, 2004; Pickel et al, 2004; Pickel et al, 2006). The ultrastructural findings of the present study suggest that the CRFr in these regions acts synergistically with glutamate largely by amplifying postsynaptic responses (Liu et al, 2004; Fu & Neugebauer, 2008), but also by enhancing glutamate release from axon terminals presynaptic to neurons that co-express the μ-OR and CRFr (Liu et al, 2004; Orozco-Cabal et al, 2008). The prevalence of the CRFr and μ-OR in the same postsynaptic dendrites of the CeL and BSTal is consistent with the respective CRFr or μ-OR facilitation and inhibition of excitatory transmission within the amygdala circuitry. The activation of the CRFr can facilitate or depress excitatory glutamatergic transmission depending on the brain region involved, and on prior stress or drug treatment (Liu et al, 2004; Rainnie et al, 2004; Orozco-Cabal et al, 2008). In the CeA, μ-OR agonists inhibit postsynaptic and presynaptic glutamatergic transmission (Zhu & Pan, 2004; Zhu & Pan, 2005). However, we rarely saw the μ-OR in terminals forming excitatory-type synapses, thus suggesting that the presynaptic inhibition may be mediated through indirect presynaptic mechanisms involving the excitatory-type terminals that express the CRFr. Alternatively, excitatory modulators (e.g. CRF) may be present in at least some of the unlabeled neurons that receive input from μ-OR-expressing terminals.

Most of the neurons in the CeA and BNST are GABAergic (Sun & Cassell, 1993), implying that many excitatory-type terminals observed in these regions are extrinsic in origin. Extrinsic excitatory input to CRFr- and μ-OR-expressing dendrites could be provided by afferents from cortical areas (Yasui et al, 1991), brainstem sensory regions such as the parabrachial nucleus (Lopez de Armentia & Sah, 2007) as well as from other amygdaloid nuclei such as the basolateral amygdala and lateral amygdala (Schmued, 1994; Zhu & Pan, 2004). Given the involvement of excitatory transmission in opiate dependence (Gonzalez et al, 1997; Bossert et al, 2004; LaLumiere & Kalivas, 2008), excitatory inputs to μ-OR and CRFr-expressing dendrites within the CeL and BSTal may play an important role in stress-induced relapse in opiate addicts (Koob & Kreek, 2007).

Chronic activation of the μ-OR results in up-regulation or sensitization of the cAMP pathway as a compensatory adaptation to opiate-induced inhibition of cAMP (Duman et al, 1988; Terwilliger et al, 1991; Nestler, 1996). Complementary functional data show that chronic, but not acute, morphine treatment sensitizes electrophysiological responses of neurons to CRF in the locus coeruleus (Xu et al, 2004). Similarly, in the CeL and BSTal, where we found extensive postsynaptic co-localization of the μ-OR and CRFr, persistent activation of the Gi-coupled μ-OR may lead to changes in the activity of the Gs-coupled CRFr. This mechanism would be equally important for enabling chronic stress to affect μOR-dependent opioid systems as well as contributing to anxiety-like states during opiate withdrawal.

Acknowledgments

We thank Drs. Gavril Pasternak, John Pintar and Sabita Roy for generous donation of brain tissue from mu-opioid receptor knockout mice.

Supporting Grants: This work was supported by NIDA (DA00727416) to AJ, DA04600 and NIMH (MH40342) to VMP, and DA016735 to Mary Jeane Kreek (The Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY.)

Abbreviations

BNST

bed nucleus of the stria terminalis

BSA

bovine serum albumin

BSTal

anterolateral bed nucleus of the stria terminalis

CeA

central amygdala

CeL

lateral central amygdala

CRF

corticotropin-releasing-factor

CRFr

corticotropin-releasing-factor receptor

CRFr-1

corticotropin-releasing-factor receptor 1

CRFr-2

corticotropin-releasing-factor receptor 2

i.p.

intraperitoneal

PB

phosphate buffer

PBS

phosphate-buffered saline

TBS

Tris-buffered saline

μ-OR

mu-opioid receptor

Footnotes

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