Research ArticleNeuroscience

Increased Abundance of Opioid Receptor Heteromers After Chronic Morphine Administration

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Science Signaling  20 Jul 2010:
Vol. 3, Issue 131, pp. ra54
DOI: 10.1126/scisignal.2000807

Abstract

The μ and δ types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the μ-δ heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of μ-δ heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the μ-δ heteromer could be a therapeutic target in the treatment of chronic pain and addiction.

Introduction

Morphine, the choice analgesic in the treatment of chronic pain, elicits its effects through opioid receptors. Repeated or continuous use of morphine leads to the development of tolerance and physical dependence. Opioid receptors are members of the heterotrimeric guanosine 5′-triphosphate–binding protein (G protein)–coupled receptor (GPCR) superfamily characterized by the presence of seven transmembrane regions. To date, three subtypes of the opioid receptor have been identified: μ, δ, and κ. Functional and physical interactions between these receptor subtypes have been noted (15). Heteromerization between μ and δ opioid receptors leads to distinct receptor pharmacology in that doses of δ receptor ligands (agonists and antagonists) too low to trigger signaling can potentiate the binding and signaling of μ receptor agonists, an effect not seen in cells expressing only μ receptor homomers (6, 7). In addition, although homomers of μ or δ opioid receptors signal through pertussis toxin–sensitive inhibitory G proteins, Gαi, the μ-δ heteromer either couples to a pertussis toxin–insensitive G protein, Gαz (8), or exhibits a switch in receptor coupling from G protein to β-arrestin2 (9, 10). In addition, μ-δ heteromerization could play a role in morphine-mediated analgesia because the analgesic effects of morphine are mediated through μ receptors (11). Moreover, low doses of δ receptor antagonists can potentiate morphine-mediated analgesia (7). For these reasons, μ-δ heteromers are considered to be a choice target for the development of new therapies to treat chronic pain (12). However, relatively little information is available about the biochemical and signaling properties of the endogenous heteromers and their regulation under pathological conditions, mainly as a result of the lack of appropriate tools to study heteromers in situ.

In the case of GPCRs, antibodies have been used as tools for receptor characterization, as reagents for their purification and tissue localization, and as probes for mapping their functional domains (13). We reasoned that heteromer-specific antibodies would be a useful tool to study endogenous heteromers in tissue, to probe their regulation in situ, and to delineate the mechanisms of regulation. Using a subtractive immunization strategy (1316) in which antibody-producing cells to unwanted antigens are eliminated through cyclophosphamide treatment, leading to the enrichment of cells producing antibodies to the desired antigen (in this case, a region shared by the heteromer), we generated μ-δ heteromer–selective antibodies. Using these heteromer-selective antibodies, we show that conditions that lead to the development of morphine tolerance correlate with increased abundance of the μ-δ heteromer in regions of the brain involved in pain perception. This suggests that the μ-δ heteromer could play a role in the development of morphine tolerance. Because the μ-δ heteromer exhibits unique pharmacology in that low non-signaling doses of δ receptor ligands can potentiate μ receptor–mediated binding and signaling, as well as morphine antinociception (610), these results identify this heteromer as a target for the development of new therapeutics in the treatment of chronic or neuropathic pain.

Results

Generation of μ-δ heteromer–selective antibodies

We used a subtractive immunization strategy (14) to generate antibodies that selectively recognize the endogenous μ-δ heteromer but do not recognize either μ or δ receptors (table S1). Mice were first made tolerant to unwanted epitopes on membrane proteins by the simultaneous administration of human embryonic kidney 293 (HEK293) cell membranes and cyclophosphamide, which causes the destruction of antibody-generating activated B cells (1416). Once a low titer to HEK293 membrane proteins was achieved, mice were immunized with membranes from HEK293 cells coexpressing μ-δ receptors (fig. S1A). The spleens of mice with high antibody titers were used to generate monoclonal antibodies. The supernatants from the resultant hybridoma clones were screened with HEK293 membranes alone, membranes from cells expressing only μ or δ receptors, and membranes from cells coexpressing both μ and δ receptors. This led to the identification of various antibody-secreting clones (table S1), including the 1E12D1 clone that gave a high signal with membranes from cells coexpressing μ and δ receptors, but not with membranes from cells expressing only μ or δ receptors (table S1).

The 1E12D1 antibody–secreting clone recognized an epitope in membranes from wild-type animals, but not from animals lacking μ or δ receptors, and in cells coexpressing μ and δ receptors, but not in cells coexpressing μ or δ receptors in combination with other GPCRs (Fig. 1, A to C, and fig. S1B). Preincubation of the antibody with membranes from HEK293 cells expressing both μ and δ receptors, but not those from cells expressing the receptors individually, substantially decreased the recognition of an epitope in SK-N-SH cells, presumably the endogenous μ and δ receptors (fig. S1C). In addition, the μ-δ heteromer antibody exhibited maximal recognition when μ and δ receptors were expressed at a 1:1 ratio, but not at a 5:1 or a 1:5 ratio (fig. S1D). Furthermore, the antibody recognized wild-type μ and δ receptors better than μ-δ chimeric constructs (table S2). These results indicate that the 1E12D1 monoclonal antibody exhibited μ-δ heteromer selectivity. Using these heteromer-selective antibodies, we could isolate μ-δ heteromers from cells expressing recombinant μ-δ receptors, as well as endogenous μ-δ heteromers from primary dorsal root ganglion (DRG) neurons (fig. S1E).

Fig. 1

Detection of μ-δ heteromers with heteromer-selective monoclonal antibodies (Ab). (A) Receptor abundance was determined in cortical membranes from wild-type (WT), μ knockout (KO), δ KO, or μ-δ double-KO mice with monoclonal antibodies to μ-δ, μ, or δ receptors by ELISA. (B and C) Cells endogenously (B) or stably (C) coexpressing μ-δ receptors were treated with or without morphine (1 μM) for 48 hours. Heteromer abundance was determined by ELISA with μ-δ heteromer–selective antibodies. Results are means ± SEM (n = 3 experiments). *P < 0.05. (D to G) Repeated morphine treatment increased μ-δ heteromer immunoreactivity in MNTB. Immunoreactivity in μ KO or δ KO mice after morphine treatment was below the detection limits. OR, opioid receptor. (H) Individual neuronal profiles were outlined (n = 15 to 20 per group) and μ-δ heteromer immunoreactivity, as assessed by mean optical density (OD), was determined. Morphine treatment increased μ-δ heteromer immunoreactivity in MNTB neurons from WT mice (**P < 0.01 relative to untreated control), but not in those from μ or δ KO mice.

Increased abundance of endogenous μ-δ heteromers after chronic morphine treatment

Because chronic opiate treatment has been reported to lead to increased opioid receptor abundance (17), we examined the effect of chronic morphine treatment on the heteromerization and subcellular distribution of μ and δ opioid receptors in cultured DRG neurons. Immunofluorescent labeling of cultured DRG neurons with antibodies to μ and δ opioid receptors showed μ-δ colocalization in neurons after vehicle or prolonged morphine treatment (fig. S2A). Increased abundance and colocalization was detected at the plasma membrane, as well as in intracellular compartments. Quantification of the extent of colocalization of the two receptors demonstrated significantly increased probability of μ and δ heteromer formation in morphine-treated DRG neurons relative to that of vehicle-treated controls (fig. S2B) in all cell sizes. Chronic, but not acute, treatment led to a significant increase in μ-δ heteromer immunoreactivity that was also detected by enzyme-linked immunosorbent assay (ELISA; fig. S2, B and C).

Next, we determined whether chronic morphine treatment triggers the formation of μ-δ heteromers using a chronic escalating dose of morphine administration paradigm (18) that leads to the development of antinociceptive tolerance. Immunohistochemistry with the μ-δ heteromer–selective antibody revealed increased μ-δ heteromer immunoreactivity after morphine administration in the medial nucleus of the trapezoid body (MNTB), an auditory relay nucleus (Fig. 1, D to G), and the rostral ventral medulla (RVM), a key relay nucleus of pain perception (fig. S3, A to E), relative to saline treatment in wild-type mice. The Allen Brain Atlas (http://www.brainatlas.org/), a genome-wide map of gene expression in the mouse brain created by high-throughput in situ hybridization analysis, shows that messenger RNAs (mRNAs) encoding the μ and δ receptors are abundant in these nuclei. μ-δ Heteromer immunofluorescence in MNTB neurons was higher in wild-type mice than in mice lacking either μ or δ receptors (Fig. 1, D to H). In MNTB neurons, μ-δ heteromer immunoreactivity was detected in putative glycinergic neurons that stained for parvalbumin, a calcium-binding protein found in neurons that use either γ-aminobutyric acid or glycine (or both) as neurotransmitters (1921) (fig. S3, F to I). A single (acute) administration of morphine (30-min treatment) did not significantly alter the abundance of μ-δ heteromer immunoreactivity in either MNTB or RVM as determined by immunohistochemistry (Fig. 2, A to K) or ELISA (Fig. 2L). In contrast, chronic escalating morphine treatment induced μ-δ heteromer formation, as indicated by both the significantly increased density and the intensity of μ-δ heteromer immunoreactivity in MNTB neurons (Fig. 2, A to K). Within MNTB, immunoreactivity was localized to glycinergic neurons ensheathed by perineuronal nets, a specialized form of extracellular matrix that provides active neurons with a well-hydrated, strongly anionic microenvironment that facilitates fast-spiking neuronal activity and the rapid transport of cations (22) (Fig. 2, A to K). Overall, our results indicate that chronic morphine treatment selectively increases μ-δ heteromer assembly in brainstem neurons.

Fig. 2

Chronic morphine treatment induces μ-δ heteromer abundance in different brain regions. (A to D) Chronic (5 days of escalating dose regime), but not acute (30 min), morphine treatment significantly increased μ-δ heteromer immunoreactivity in perineuronal net-bearing MNTB neurons detected by WFA. Arrowheads indicate neurons bearing μ-δ immunoreactivity that are ensheathed by perineuronal nets. Scale bars, 25 μm. (D) Mean grayscale optical density measured within individual neurons of two to four mice. (E to K) Quantitative analysis of the maximal fluorescence intensity of μ-δ immunoreactivity in individual perineuronal net-bearing neurons of MNTB after acute or chronic morphine treatment. Numbers in parentheses indicate the number of neurons. Scale bars, 18 μm. **P < 0.01. (L) ELISA with membranes from MNTB, RVM, or cortex shows that a 5-day chronic escalating dose of morphine but not a single dose of morphine (acute, 10 mg/kg) or saline significantly increased μ-δ receptor abundance. Results are means ± SEM (n = 3 experiments). ***P < 0.001 relative to saline. (M) ELISA with membranes from different mice brain regions shows that treatment with morphine (5 mg/kg) or NTB (0.1 mg/kg) for 9 days significantly increased μ-δ receptor abundance. Results are means ± SEM (n = 3 experiments). *P < 0.05; **P < 0.01; ***P < 0.001. Hippoc, hippocampus; Hypoth, hypothalamus, N.Acc., nucleus accumbens; PFC, prefrontal cortex; VTA, ventral tegmental area.

To quantify the morphine-induced increase in abundance of μ-δ heteromers, we carried out ELISA assays with brain membranes from saline- and morphine-treated animals. Animals treated chronically with morphine showed increased heteromer abundance in the cortex, MNTB, and RVM (Fig. 2L), as well as in the hypothalamus, nucleus accumbens, and ventral tegmental area, relative to saline-treated controls (Fig. 2M). We have previously shown that δ antagonists can potentiate morphine analgesia. Therefore, we examined whether chronic treatment with naltriben (NTB), an alkaloid δ antagonist, enhances μ-δ heteromer abundance. Similar to chronic morphine treatment, chronic treatment with NTB caused increased μ-δ heteromer abundance in different brain regions (Fig. 2M). In addition, treatment of cells expressing recombinant or native μ and δ receptors with morphine for 48 hours significantly increased the abundance of the μ-δ heteromer (Fig. 1, B and C and fig. S4B). Thus, a component of the morphine-mediated up-regulation of μ-δ heteromer formation can be recapitulated in cell culture systems. Therefore, we used Chinese hamster ovary (CHO) cells expressing recombinant μ and δ receptors and SK-N-SH cells expressing native μ and δ receptors to determine whether only morphine induced increased μ-δ heteromer abundance. Treatment of CHO cells for 48 hours with alkaloid but not peptide ligands (either antagonists or agonists) increased μ-δ heteromer abundance (Fig. 3A). The time course analysis in SK-N-SH cells revealed that this increase was seen within 2 hours of treatment and peaked around 8 hours (Fig. 3B). This increase was also seen in cells expressing recombinant receptors, thereby suggesting a posttranslational mode of regulation of the heteromer.

Fig. 3

Chronic alkaloid treatment increases μ-δ heteromer abundance. (A) CHO cells stably expressing μ and δ receptors were treated with the indicated ligands (1 μM) for 48 hours and μ-δ heteromer abundance was determined by ELISA. Results are means ± SEM (n = 3 to 4 experiments). **P < 0.01. (B) Morphine-treated (1 μM) SK-N-SH cells were probed by ELISA with monoclonal antibodies to μ, δ, or μ-δ receptors. Results are means ± SEM (n = 3 to 4 experiments).

Inhibition of agonist binding and signaling by the μ-δ heteromer–selective antibody

We examined the ability of the μ-δ heteromer–selective antibody to selectively inhibit the ability of low doses of the δ antagonist TIPPψ [(2S)-2-[[(2S)-2-[[(3S)-2-[(2S)-2-amino-3-(4-hydroxyphenyl)propanoyl]-3,4-dihydro-1H-isoquinolin-3-yl]methylamino]-3-phenylpropanoyl]amino]-3-phenylpropanoic acid] to potentiate the binding and signaling of the μ agonist DAMGO (Tyr-d-Ala-Gly-MePhe-Gly-ol) (6, 7). The μ-δ heteromer–selective antibody significantly decreased δ antagonist–mediated increases in the binding of μ agonist (Fig. 4A and fig. S5A). We used the [35S]guanosine 5′-O-(3′-thiotriphosphate) ([35S]GTP-γ-S) binding and adenylyl cyclase activity assays to measure the effect of the heteromer-selective antibody on signaling events downstream of receptor activation. Activation of opioid receptors leads to exchange of guanosine diphosphate (GDP) for guanosine 5′-triphosphate (GTP) at the associated G protein (Gαi) subunit; this can be measured with a radiolabeled nonhydrolyzable analog of GTP, [35S]GTP-γ-S. This exchange, in turn, leads to the activation of the Gαi subunit followed by inhibition of adenylyl cyclase activity and, consequently, decreases in intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) concentrations. Using these signaling assays, we found that the heteromer antibody significantly blocked the δ antagonist–mediated increases in μ receptor agonist–mediated signaling in membranes from wild-type animals, but not those from animals lacking μ or δ receptors (Fig. 4, B and C, and fig. S5, B to D).

Fig. 4

μ-δ Heteromer–selective antibody blocks heteromer-mediated increases in binding and signaling. (A) CHO cells coexpressing Flag-tagged μ and Myc-tagged δ receptors were pretreated with either the μ-δ heteromer–selective antibody or monoclonal antibodies to Flag or Myc. Binding of the μ receptor agonist [3H]DAMGO (10 nM) to cells was measured in the presence or absence of the δ receptor antagonist TIPPψ (10 nM). Results are means ± SEM (n = 3 experiments). (B and C) Mouse cortical membranes were preincubated with or without μ-δ heteromer antibodies and G protein activity, as assessed by [35S]GTP-γ-S binding (expressed as G protein activity) (B), or adenylyl cyclase activity (C) in response to DAMGO (1 μM) was determined in the presence or absence of TIPPψ (10 nM). Results are means ± SEM (n = 3 experiments). (D) HEK293 cells coexpressing a G16-Gi3 chimera and μ and δ opioid receptors were preincubated with or without μ-δ heteromer antibodies, and then treated with the δ opioid agonist deltorphin II (1 μM) in the presence or absence of the μ opioid antagonist CTOP (10 nM), and intracellular Ca2+ concentrations were determined. Results are means ± SEM (n = 3 experiments). *P < 0.05; **P < 0.01; ***P < 0.001.

We have previously shown that μ receptor antagonists can potentiate δ agonist binding and signaling in cells coexpressing μ and δ receptors (6). To determine whether the heteromer-selective antibody could block μ antagonist–mediated increases in δ agonist signaling, we used an assay in which cells are transfected with μ and δ receptors along with a chimeric G16-Gi3 protein. The activation of this chimeric G protein after binding of a receptor agonist results in increases in intracellular Ca2+ concentrations that can be measured with a Ca2+-binding dye. We found that the heteromer-selective antibody blocked μ-δ heteromer–mediated [deltorphin II, a δ agonist, in the presence of CTOP (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2), a μ antagonist] increases in intracellular Ca2+ concentrations in cells coexpressing these receptors (Fig. 4D). These results indicate that our μ-δ heteromer antibody exhibits receptor heteromer selectivity and can be used to probe μ-δ heteromer–mediated effects.

Discussion

Various in vitro studies have demonstrated that the heteromerization of GPCRs alters their pharmacological and functional properties (15). However, the evaluation of the in situ distribution of heteromers—and their functional relevance and modulation under pathological conditions—has been hampered by the lack of appropriate tools. We generated heteromer-selective antibodies using a subtractive immunization strategy in which cyclophosphamide was used to kill cells that generated antibodies to undesired antigens (in this case, HEK293 membranes), thereby increasing the exposure of the desired antigen (μ-δ heteromers) to antibody-producing cells. Our heteromer-selective antibody enabled the detection and isolation of μ-δ heteromers and blocked heteromer-mediated signaling. Thus, a subtractive immunization strategy could be used to generate antibodies against other GPCR heteromers and enable studies examining the abundance and regulation of receptor multimers in normal and pathological states.

The heteromer-selective antibody generated in this study blocked heteromer-mediated ligand binding and signaling and hence could be used to determine the biological contribution of heteromers compared to homomers after receptor activation. These findings are relevant to opiate action because the relative ratio of μ-δ heteromers to μ homomers could play a role in modulating the response to an opiate, and studies have shown that the heteromer transduces signaling through a pathway that is distinct from that of receptor homomers (9). μ-δ Receptors display decreased G protein coupling and signaling relative to that of μ homomers and exhibit a β-arrestin–mediated μ receptor response (9). This, in turn, leads to changes in the spatiotemporal dynamics of signaling, as assessed by the phosphorylation status and localization of extracellular signal–regulated kinase (ERK). In cells expressing μ homomers, ERK shows peak phosphorylation at 3 to 5 min after stimulation and the activated kinase localizes primarily to the nucleus, whereas in cells expressing μ-δ heteromers, ERK shows peak phosphorylation at 15 min and the activated kinase is retained in the cytoplasm (9). This causes differential activation of transcription factors (9) that could be responsible for the differences in gene expression that occur with the development of morphine tolerance. Thus, the switch to the “β-arrestin–dependent” signaling cascade could contribute to the changes in morphine response that underlie tolerance. In addition, the absence of morphine tolerance in animals lacking δ receptors (23) or in animals with reduced surface abundance of δ receptors resulting from deletion of the gene encoding preprotachykinin (24) is consistent with a requirement of μ-δ heteromer–mediated signaling in the development of morphine tolerance. Furthermore, mouse knockouts lacking β-arrestin2 or δ receptors do not develop morphine tolerance (23, 25). Thus, μ-δ heteromers, through their association with β-arrestin2, could contribute to the development of tolerance to morphine. Although our data showing increased μ-δ heteromer abundance after chronic morphine administration would support this notion, further studies are needed to examine how interactions between β-arrestin2 and μ-δ heteromers contribute to the development of morphine tolerance.

We found that μ-δ heteromer abundance was increased in RVM after chronic morphine treatment. RVM, a brain region containing both μ and δ opioid receptor mRNAs, is involved in antinociception through facilitation of the descending inhibitory pain pathways. Malfunction in this circuitry is thought to play a role in neuropathic pain, a condition characterized by the presence of hyperalgesia (supersensitivity to painful stimuli) and tactile allodynia (painful sensation by normally nonpainful stimuli) (26, 27). Therefore, if neuropathic pain increases the likelihood of μ-δ heteromer formation in RVM, this could account for the lack of analgesic potency of morphine in the treatment of neuropathic pain (28) because the μ-δ heteromer signals through β-arrestin2 (9) and deletion of β-arrestin2 gene in mice leads to a potentiation, as well as a prolongation of the analgesic effects of morphine (29). Therefore, further studies are needed to examine the effects of neuropathic pain on μ-δ heteromers in RVM. Glycinergic neurons of MNTB, an auditory relay nucleus, also showed increased μ-δ heteromer abundance after chronic morphine administration. However, not much is known about the role of these receptors in auditory processing. Because chronic morphine administration causes increased μ-δ heteromer abundance in MNTB, studies are needed to examine the role of these receptors in acute and chronic pain states.

We observed colocalization of μ and δ receptors in cultured DRG neurons that is increased upon prolonged treatment with morphine. In a study using mice with a knock-in of δ opioid receptor tagged with enhanced green fluorescent protein (δEGFP), immunostaining with antibodies against GFP and μ receptors showed that δEGFP colocalizes with μ opioid receptors in less than 5% of DRG neurons (30). This degree of colocalization could be an underestimate because these mice show increased abundance of δ opioid receptors (31), and the GFP antibody exhibits higher avidity for GFP than the μ antibody does toward μ receptors. This may result in an overestimation of δ opioid receptor abundance relative to that of μ receptors. In addition, the GFP tag at the C terminus increases the cell surface localization of the δ opioid receptor (32). Together with the evidence that increased abundance of δ opioid receptor attenuates the maturation of the μ opioid receptor (33), these results suggest that the low degree of colocalization between δEGFP and the μ opioid receptor (30) could be a result of alterations in δ opioid receptor maturation. Thus, as supported by our immunostaining data, μ and δ opioid receptors may colocalize in DRGs as well as in other regions of the brain.

In summary, we report the generation of a μ-δ heteromer–selective antibody that enabled us to examine chronic morphine treatment–mediated up-regulation of μ-δ heteromers in endogenous tissue. The subtractive immunization strategy used in the generation of the μ-δ heteromer–selective antibodies could be used to generate antibodies selective for other GPCR heteromers, which would be useful in studies examining the role of GPCR heteromers in physiological and pathophysiological conditions.

Materials and Methods

ELISAs

ELISAs were carried out as previously described (34) either with cells (2 × 105 per well) expressing individual receptors or with cells coexpressing μ and δ opioid receptors that were treated with 1 or 10 μM different opioid ligands for 0 to 72 hours, with cultured DRG neurons, or with membranes (10 μg) prepared from different brain regions of wild-type, μ knockout, δ knockout, and μ-δ double-knockout mice or from mice that were treated either acutely or with escalating doses of morphine. Data obtained with μ-δ heteromer–selective antibodies in ELISA assays are expressed as μ-δ abundance in the figures.

Binding assays

CHO or SK-N-SH cells (2 × 105 per well) coexpressing μ and δ opioid receptors were incubated with 5 μg of antibodies for 10 min at room temperature (RT), and then with 10 nM [3H]DAMGO in the presence of antibodies and in the presence or absence of 10 nM TIPPψ (35).

GTP-γ-S binding assays

Membranes (10 μg) were incubated with 5 μg of antibodies for 10 min at RT, and then with 1 μM DAMGO in the presence or absence of 10 nM TIPPψ and in the presence of antibodies. [35S]GTP-γ-S binding was determined as described (7).

Adenylyl cyclase assays

Membranes (10 μg) were incubated with 5 μg of antibodies for 10 min at RT, and then with 1 μM DAMGO in the presence or absence of 10 nM TIPPψ and in the presence of the antibodies. Adenylyl cyclase activity was determined as described (36, 37).

i16-facilitated Ca2+ release

CHO cells coexpressing a chimeric G16-Gi3 protein and μ and δ opioid receptors were plated onto poly-l-lysine–coated, 96-well clear-bottom plates (40,000 per well). The next day, the growth medium was removed, and the cells were washed twice in Hanks’ balanced salt solution containing 20 mM Hepes. Cells were incubated with Fluo-4 NW calcium dye (3 μM in 100 μl) for 1 hour at 37°C. The cells were preincubated for 30 min with or without 5 μg of antibodies. Deltorphin II (1 μM) or CTOP (10 nM) was added to the wells (in the presence of the antibodies) by the robotic arm of the FLEX Station, and intracellular Ca2+ concentration was measured for 300 s at excitation and emission wavelengths of 494 and 516 nm, respectively.

Immunoprecipitation and Western blotting

CHO cells coexpressing hemagglutinin-tagged μ and Flag-tagged δ receptors or DRGs with endogenous μ and δ receptors [isolated from embryonic rat pups at embryonic day 16 (38)] were immunoprecipitated with the μ-δ heteromer–selective antibody as described (7). μ and δ receptors were detected in the immunoprecipitates by means of polyclonal antibodies to the epitope tags (CHO cells) or to the individual receptors (DRGs). The δ receptor antibodies were from Chemicon or Proteimax, and the μ receptor antibodies were a gift from T. Cote [Uniformed Services University of the Health Sciences (USUHS)].

Colocalization of μ, δ, and μ-δ receptors in DRG neurons

DRG neurons from adult rats were grown in culture for 4 days. They were then treated with either vehicle (saline) or morphine (10 μM, 48 hours) before fixation with 4% paraformaldehyde in 0.1 M phosphate buffer for 15 min at 37°C. Immunocytochemical labeling for μ and δ opioid receptors was accomplished with antibodies against the μ receptor (Neuromics) and the δ receptor (Alamone) and Alexa 488– and Alexa 594–conjugated secondary antibodies, respectively. Photomicrographs were captured by a Leica TCS SP2 multiphoton confocal microscope (63× primary magnification; Leica Microsystems), and images were acquired and digitized for quantitative analysis with Leica Confocal Software.

Animal studies

Animal studies were carried out according to protocols approved by the Mount Sinai School of Medicine Animal Care and Use Committee. Mice lacking μ opioid (35) (n = 6) or δ opioid receptors (36) (n = 6) were back-crossed onto a C57Bl6/J background, bred, and maintained as described (39, 40). Wild-type littermates (n = 8) were used as controls in pharmacological challenge experiments. Morphine was systemically administered according to a chronic intermittent escalating dose protocol (from 10 mg/kg on day 1 to 100 mg/kg on day 5) or acutely (a single injection of 10 mg/kg). For tissue collection, each mouse brain was placed in a mouse brain mold (Braintree Scientific) and the brainstem was coronally sliced at 1-mm thickness. The ventral portion of the 1-mm slice located between −4.60 and −5.60 mm relative to bregma included MNTB and RVM. Bilateral MNTB and midline RVM regions were punched with a 14-gauge tissue needle (Scientific Commodities), pooled, and stored at −80°C until use. For immunohistochemistry, animals were perfused with ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) that was preceded by a short prerinse with physiological saline. Tissues were cryoprotected in 30% sucrose in phosphate buffer.

Immunohistochemistry

Coronal sections of wild-type and knockout mice treated or not with morphine were prepared on a cryostat microtome at a 16-μm thickness and thaw-mounted onto fluorescence-free glass slides. Sections at identical anterior-posterior coordinates from wild-type and μ or δ knockout mice treated or not with drug were mounted onto each glass slide to allow simultaneous histochemical processing. Similar simultaneous tissue processing was performed when comparing the effects of acute and chronic morphine treatments, with sections spanning MNTB and RVM, from a control, acute, or chronic morphine-treated mouse tissue mounted onto each glass slide. Specimens were rinsed in 0.01 M phosphate-buffered saline (PBS; pH 7.4), blocked in PBS containing 10% normal donkey serum and 0.3% Triton X-100, and incubated in diluted (>1:4000) supernatant from the 1E12D1 hybridoma secreting clone (expressing monoclonal μ-δ heteromer–selective antibody) that was diluted in PBS–0.3% Triton X-100 and 0.1% NaN3 at 4°C for 16 to 24 hours. Immunoreactivity was visualized with the tyramide signal amplification system (TSA-Plus; NEN Life Science Products): Sections were washed in TNT buffer [0.1 M tris-HCl (pH 7.5), 0.15 M NaCl, and 0.05% Tween 20] for 10 min, followed by 30 min of incubation with horseradish peroxidase–conjugated donkey immunoglobulin G against mouse (1:500; Jackson ImmunoResearch). After three washes in TNT, sections were exposed to fluorescein- or carbocyanine 3 (Cy3)–conjugated biotinyl tyramide diluted in amplification diluent (1:100) for 15 min at RT and thereafter washed twice in TNT buffer. After single staining to reveal μ-δ heteromers, sections were repeatedly washed in PBS and exposed to rabbit antibody against golgin subfamily A5 (GOLGA5; 1:1000; Atlas Antibodies) (3941) or goat antibody against parvalbumin (1:11,000; Swant); biotinylated Wisteria floribunda agglutinin (WFA) was used to visualize perineuronal nets (20 μg/ml; Sigma) (38) overnight at 4°C. After repeated rinses in TNT buffer, immunoreactivities were revealed by Cy2-, Cy3-, or Cy5-conjugated donkey antibodies against rabbit or goat (1:300; Jackson ImmunoResearch). WFA binding was revealed by Cy2-tagged streptavidin (1:5000; Jackson).

Specimens were imaged with a confocal laser-scanning microscope (model 510, Zeiss) equipped with appropriate excitation and emission filters for maximum separation of Cy2 (505 to 530 nm, band pass), Cy3 (560 to 610 nm, band pass) and Cy5 signals (>650 nm, long pass). Images were taken with identical pinhole, detector gain and offset, and amplification gain settings to allow direct comparisons of degree of immunoreactivity, which was measured from primary unmodified confocal laser-scanning microscope images with National Institutes of Health ImageJ optical software (version 1.41). The mean grayscale optical density and the maximum of pixel intensity were measured within individual neurons obtained from two to four animals per treatment group. Results were analyzed with one-way analysis of variance (ANOVA) with Bonferroni post hoc correction. Digital images were color-coded for optimal visualization of signals and underwent linear optimization of brightness and contrast with Adobe Photoshop CS3 (Adobe Systems).

Acknowledgments

Acknowledgments: We thank T. Cote (USUHS) for the gift of polyclonal antibodies to μ opioid receptors; B. Kieffer (Institut de Génétique et de Biologie Moléculaire et Cellulaire) for the gift of μ and δ knockout animals; J. Pintar for the gift of brains from μ, δ, and μ-δ knockout animals; A. Prodhen for help in processing the tissues; and K. Gagnidze for help in the preparation of primary DRG cultures. Funding: This work was supported in part by NIH grants DA023214 (T.H.), DC08301 (E.M.), DC06696 (E.M.), DC07984 (E.M.), T32GM062754 (I.B.), DA019521 (L.A.D.), DA08863 (L.A.D.), and GM071558 (L.A.D.); Canadian Institutes of Health Research and Canada Research Chairs (C.M.C.); Natural Sciences and Engineering Research Council of Canada (E.O.); the Scottish Universities Life Science Alliance (T.H.); and the European Molecular Biology Organization Young Investigator Program (T.H.). J.M. is an Alzheimer’s Research Trust fellow. Author contributions: A.G. generated heteromer-selective monoclonal antibodies and carried out ELISA and binding assays; J.M. carried out immunohistochemistry studies with animal tissue; I.G. carried out Western blot analysis, signaling assays, and data analysis and helped in manuscript preparation; R.R. helped with signaling assays and critically reviewed the manuscript; I.B. carried out ELISA assays with MNTB and RVM membranes; E.O. examined the effect of morphine on heteromer abundance in DRG cultures; M.L. carried out reverse transcription polymerase chain reaction studies; E.M. helped with intracellular calcium assays; M.J. helped with experiments examining the effect of morphine on heteromer abundance in DRG cultures; C.M.C. designed and analyzed immunofluorescence experiments with DRGs; T.H. designed and analyzed immunohistochemistry data with animals; and L.A.D. initiated this project, designed the experiments, and wrote the manuscript. Competing interests: The authors declare that they have no competing interests.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/3/131/ra54/DC1

Materials and Methods

Fig. S1. Generation and characterization of μ-δ heteromer–selective antibodies.

Fig. S2. Subcellular distribution and colocalization of μ and δ opioid receptors.

Fig. S3. Chronic morphine treatment induces increased μ-δ heteromer immunoreactivity in the brainstem.

Fig. S4. Chronic morphine treatment increases μ-δ heteromer abundance.

Fig. S5. μ-δ heteromer–selective antibodies block heteromer-mediated binding and signaling.

Table S1. Characterization of select hybridoma clones.

Table S2. Heteromer-selective antibody recognition using chimeric μ-δ constructs.

References

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