The fluorescent naltrexamine-acylimidazole was designed based upon the crystal structure of β-funaltrexamine (β-FNA) bound MOR (Manglik et al., 2012). The key covalent attachment of β-FNA at K233 side-chain was purposely avoided. The linker for NAI was designed to be longer than that for β-FNA and was predicted to chemically modify nucleophilic side chains of amino acids on the extracellular loops 2 and 3 (Figure 1—figure supplement 1). The compound was made in two steps. First, naltrexamine-acylimidazole alkyne (NAI-AK) was synthesized. The β-epimer of naltrexamine was stereospecifically derived from a non-selective opioid antagonist naltrexone (Sayre and Portoghese, 1980). Acylimidazole was incorporated into the naltrexamine to produce a proper linker and a terminal alkyne (Figure 1B). The second part of the synthesis was designed for coupling an azide probe to NAI-AK via copper-catalyzed azide-alkyne cycloaddition known as click chemistry (Figure 1C; Tornøe et al., 2002; Rostovtsev et al., 2002). The click reaction was optimized to be in slightly acidic conditions (pH≈4) to prevent hydrolysis of the acylimidazole linker. Incorporation of Alexa 594 azide to NAI-AK gave NAI-A594 (Figure 1C) with a good yield and high purity following high performance liquid chromatography. Competitive radioligand binding assays showed that NAI-A594 bound to MOR, DOR and KOR with Ki ≈ 50, 70 and 200 nM, respectively (n = 2 of triplicate samples, see Table 1). NAI-A488 was prepared as described for NAI-A594.

HEK293 cells stably expressing FLAG-epitope tagged MORs (FMOR) were first used to test the ability of NAI-A594 to transfer the fluorophore to the receptor. In the first experiment, the stability of NAI-A594 labeling on living cells was visualized with a spinning disc confocal microscope. FMOR-expressing cells were labeled with NAI-A594 and as a control, co-labeled with anti-FLAG (M1) antibody conjugated to Alexa 488 (M1-A488). Confocal images revealed the fluorescence of A594 and M1-A488 along the plasma membrane even after washing with the high affinity antagonist, naloxone (Figure 2A–a). For comparison, the reversible fluorescent ligand, naltrexamine-Alexa594 (NTX-A594, K_d ~53 nM, Birdsong et al., 2013) was used for labeling instead of the NAI-A594. The fluorescence of A594 but not of M1-A488 on plasma membrane was almost completely lost after the naloxone-wash (Figure 2A–b).

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Given that NAI-A594 also binds to DOR and KOR in nanomolar concentrations, experiments using FLAG-DOR (FDOR, stably expressed) and FLAG-KOR (FKOR, transiently expressed) in HEK293 cells were done (Figure 2—figure supplement 1). After washing with naloxone (1 µM) the fluorescence of NAI-A594 on FDOR expressing cells was comparable to that in FMOR expressing cells. Persistent labeling of FKOR was negligible.

Next, flow cytometry was used to confirm in FMOR cells that NAI-A594 efficiently labels the receptors. The average fluorescence intensity of cells labeled with NAI-A594 (100 nM, 1 hr at 37°C) was similar to antibody labeling using M1-A594 despite the fact that antibodies had an average four dye molecules per protein. Application of naloxone (10 µM) decreased the average fluorescence intensity by 30%, suggesting that there was a reversible component of NAI-594 labeling (Figure 2B). When naloxone was co-incubated during the labeling of NAI-A594 (100 nM), the average fluorescence intensity was reduced to only 2% of the NAI-A594 treatment alone, indicating that non-specific staining was negligible.

Under the same conditions the fluorescence intensity of cells labeled with the non-covalently labeling probe NTX-A594 (100 nM) was examined. The fluorescence intensity was only 40% of that with NAI-A594 and was further reduced with the application of naloxone. As the concentration of NAI-A594 increased, the fluorescence labeling also increased (Figure 2B).

Biochemical experiments were also carried out to verify that the dye moiety was in fact irreversibly bound to the receptors. In one set of experiments, NAI-A488 was synthesized, and used to label cells for subsequent immunoprecipitation and Western blot analysis (Figure 2C). In brief, FMOR cells were incubated with NAI-A488 (100 nM) in the absence (lane 4) and presence of naloxone (lane 3). Unlabeled FMOR cells (lane 2) and HEK293 cells not expressing FMOR (lane 1) were also included in the assays. Next, cell extracts were prepared and FMOR was immunoprecipitated using a rabbit anti-MOR antibody (UMB3) followed by western blotting with either rabbit anti-A488 (Figure 2C, left gel) or anti-FLAG mouse monoclonal (M1, Figure 2C, right gel). Immunoblotting with anti-A488 detected a ~ 75 kDa band of FMOR only in cells labeled with NAI-A488 in the absence of naloxone (Figure 2C, left gel, lane 4), indicating that A488 was irreversibly bound to FMOR. In contrast, the anti-FLAG antibody detected FMOR in all treatment conditions except the control HEK293 cells, verifying that FMOR was present in unlabeled as well as labeled cells. (Figure 2C, right gel, lanes 2–4). Nonspecific bands near 35 and 100 kDa were observed in every sample including the control HEK293 cells (Figure 2C, lane 1–4 of left and right panels). The fluorescence spectra of the cell extracts used in this experiment confirmed the results from the immunoprecipitation and Western blot analysis and displayed maximal emission at 517 nm, characteristic of A488 dye (Figure 2D). Fluorometry also confirmed that labeling of cells with NAI-A594 was FMOR-specific, and the emission maximum of 615 nm was consistent with the A594 fluorophore (Figure 2—figure supplement 2A).

The NAI compound was also used to directly label purified receptors. In this experiment, the purified FMOR (0.75 μM) was incubated with NAI-A488 (4.7 μM) at room temperature in the presence or absence of naloxone (100 μM). Aliquots were removed at 0, 4, 24, 48, and 96 hr and denatured by addition of SDS-sample buffer. Analysis of in-gel fluorescence showed that the attachment of A488 to receptors increased with time and reached maximal intensity after 48 hr (Figure 2E). No significant fluorescence was found in preparations co-incubated with naloxone, indicating that this labeling was specific to FMOR (Figure 2—figure supplement 2B).

Agonist-induced internalization of receptor could also be followed in cells that were treated with NAI-A594. FMORs labeled with NAI-A594 were found co-localized with M1-A488 in the cytoplasm after [Met⁵]enkephalin (ME) application, indicating that the A594 labeled receptors could bind agonist, internalize, and be visualized in the endosomes (Figure 2F,a–d).

Several brain areas were selected to evaluate NAI-A594 labeling of receptors on living neurons from rats and mice, as discussed below.

Locus Coeruleus

Locus coeruleus (LC) neurons were first selected to test the labeling efficiency and functionality of the receptors after labeling. These noradrenergic neurons are an excellent model because they express only the MOR subtype and are a near homogenous population of densely packed neurons. Brain slices containing LC neurons were prepared and incubated in a solution of NAI-A594 (100 nM) in oxygenated artificial cerebrospinal fluid (ACSF) at 30°C for 1 hr. Following incubation, macroscopic images showed fluorescent signals in the area of LC (Figure 3A–a). At higher magnification, single LC neurons were identified (Figure 3A–a’). With the use of a 2-photon microscope, fluorescence of Alexa594 was observed along the plasma membrane of LC neurons and on many processes (Figure 3B–a). Fluorescence remained after application of naloxone (10 µM, 10 min) indicating that the labeling was stable (Figure 3B–a’). Quantification of cell-associated fluorescence revealed a 40% decrease in the intensity of labeling after application of naloxone (Figure 3—figure supplement 1A). In contrast, labeling with the reversible ligand, NTX-A594, was not efficient and the fluorescence along the plasma membrane was nearly abolished after washing with ACSF for 10 min (Figure 3B–b,b’). There was no staining of NAI-A594 in LC neurons from MOR knockout rats (Figure 3B–c,c’). To further confirm the specific labeling of NAI-A594, slices from mice expressing green fluorescence protein under regulation of tyrosine hydroxylase promotor (THGFP) were used as a marker for the noradrenergic LC neurons. The staining of NAI-A594 was observed on the plasma membrane of both somata and dendrites of GFP expressing LC cells (Figure 3B–d). The labeling of NAI-A594 in the GFP positive neurons was blocked when slices were co-incubated with CTAP (1 µM), a MOR-selective antagonist (Figure 3B–d’). The fluorescence puncta found in these slices are autofluorescence from natural substrates presented in the neurons. These puncta were observed in both GFP and non-GFP neurons (Figure 3—figure supplement 2).

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In the same horizontal slices that contained the LC neurons, strong fluorescent labeling with NAI-A594 was also found in the nearby lateral parabrachial nucleus (PB, Figure 3A–a). The labeling of a single neurons in this area was visualized with the 2-photon microscope (Figure 3C–a). Neurons in PB area labeled with NAI-A488 were also observed using the spinning disc confocal microscope (Figure 3C–b). Lateral to the pons, labeled neurons in hippocampal formation (HC) were also clearly visualized (Figure 3A–a). The fluorescent pattern in the hippocampus was very similar to that observed using MOR-specific immunohistochemistry in fixed tissue (Arvidsson et al., 1995a).

Labeling of endogenous MORs with NAI-A594 was both concentration- and time-dependent (Figure 3—figure supplement 1B and C). After incubation for 1 hr, labeling was observed with NAI-A594 at concentrations as low as 10 nM. Increasing the concentration to 100 nM resulted in a well-defined florescent signal along the plasma membrane. The time course of labeling was examined using NAI-A594 at a concentration of 30 nM. Incubation times from 0.5 to 2 hr showed an increase in staining. Brian slices that were left in the incubation vials longer than 2 hr did not result in additional increased fluorescence (data not shown). This rapid labeling at low concentrations makes NAI-A594 suitable for identifying receptors in living slices that will be used for functional experiments.

A distinct advantage for using the small molecule labeling approach is that the NAI compounds can penetrate and label receptors on the neurons deep into the slice, thus enabling detection of healthy neurons in three-dimensions. The penetration and labeling of the NAI-A594 was examined using brain slices from a transgenic animal expressing FMORs in LC. The same neurons were labeled with M1-A488 and NAI-A594 for comparison. LC neurons having FMORs labeled with NAI-A594 were observed deep (90 µm) in the slice at a depth where visualization of FMORs labeled with M1-A488 was not well-defined (Figure 3D–a,b, Figure 3—figure supplement 1D,a for quantification, and Video 1 and Video 2). M1-594 labeling was also investigated in LC slices and compared to separate LC slices labeled with NAI-A594. The depth of labeling using M1-A594 was similar to that of M1-A488, less than the labeling of NAI-A594. (Figure 3—figure supplement 1D,b for quantification, and Video 3 and Video 4).

Video 1

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Striatum and cerebral cortex

All three opioid receptor subtypes are found within the striatum and nucleus accumbens (Arvidsson et al., 1995b; Miura et al., 2007; Banghart et al., 2015). MOR is present predominantly in the striosomes or patches. In contrast, DOR and KOR are diffusely distributed throughout the region. In coronal slices of striatum from mice, NAI-A594 labeling revealed a distinct distribution of fluorescent patches (Figure 4-a,d). Labeling was blocked when slices were co-incubated with CTAP (1 µM, Figure 4-b) indicating that NAI-A594 primarily labeled MORs. This distinctive patch distribution was also observed in coronal and horizontal slices of wild type rats (Figure 4-c,e). Labeling was also observed at the cellular level with both a spinning disc confocal and 2-photon microscopy (Figure 4-c,f). No fluorescent structures were observed in the CTAP-treated slices examined with the spinning disc or 2-photon microscopes (Figure 4—figure supplement 1a). Perhaps surprisingly, NAI-A594 did not efficiently label endogenous DORs in brain slices of the cortex where it has been shown to have DOR immunoreactivity (Cahill et al., 2001). Using 2-photon microscopy, sparse labeling of processes was observed that was blocked in CTAP treated slices (Figure 4—figure supplement 1b).

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Thalamus and nearby structures

Coronal slices from rat and mouse thalamus were examined next. This brain area exhibits dense MOR binding sites as shown by autoradiography (Mansour et al., 1988). Strong fluorescence induced by NAI-A594 was found in the thalamus and habenula (Figure 5-a,d,e). Consistent with the results from the striatum, all the labeling was absent in brain slices from MOR knockout rats (Figure 5-b). Using a 2-photon microscope, the labeled neurons in the periventricular nucleus of the thalamus (PVT) were identified in a heterogeneous population where other unlabeled neurons were also present (Figure 5-c). Fluorescent neurons in the mouse habenula were identified (Figure 5-d,e,f), and this labeling was also blocked by CTAP (data not shown).

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Ventral tegmental area and substantia nigra

The labeling of NAI-A594 was next investigated in the substantia nigra and ventral tegmental area of the midbrain. Dopamine cells in these areas are known to express KOR (Arvidsson et al., 1995b; Ford et al., 2007). There is also a small population of dopamine neurons that are sensitive to ME and thus likely express MOR (Ford et al., 2006).

These experiments used THGFP mice to identify dopamine cells. When midbrain slices were incubated in NAI-A594, only ~3% of THGFP positive cells showed staining with NAI-A594 (Figure 6A–a,c,c’; 3 out of 90 cells, eight slices, three animals). Although THGFP negative neurons were labeled with NAI-A594, the population was not quantified (Figure 6A–a). Perhaps the most remarkable observation was the amount of Alexa594 fluorescence found on processes lacking GFP fluorescence (Figure 6A–a,d,d’). The labeling of these processes was blocked by naloxone (10 µM) and CTAP (1 µM, Figure 6B). These results underscore the complexity of opioid systems in the midbrain where inputs of MOR sensitive neurons from several brain areas are reported (Matsui et al., 2014). It is expected that NAI-A594 will afford a convenient way to study these neurons and their functionality in slices from wild type animals.

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After NAI-A594 labeling, MORs are functional

Collectively, the results from labeling studies of neurons in brain slices demonstrate that NAI-A594 reacted selectively with the MOR subtype. The next important question is to determine if NAI-A594 labeled neurons function normally. The activation of G protein-coupled inwardly-rectifying potassium conductance (GIRK) in LC neurons was used to determine NAI-A594 labeled MORs remained active. First, does NAI-A594 act as an antagonist? Brain slices containing LC neurons were prepared and whole cell recordings were made. [Met⁵]enkephalin (ME, 300 nM) caused an outward current that was reduced by half after applying NAI-A594 (500 nM, 5 min, Figure 7A, left panel). After washing NAI-A594, the ME-induced current increased toward the pretreatment control indicating that the receptors were free of antagonist. Following this experiment, the slices were removed from the recording chamber and imaged using a macroscope. A strong fluorescent signal was observed in the LC and parabrachial nucleus (PB, Figure 7A, right). The results demonstrate that after the chemical reaction of NAI-A594 with the receptors, the naltrexamine part of this compound was released and dissociated leaving functional receptors.

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In general, labeling and identifying MOR containing neurons required only 30–100 nM NAI-A594. To examine the function of the neurons following a typical labeling procedure, whole cell recordings were done with LC slices from wild type rats that had been incubated with NAI-A594 (30 nM, 1 hr). In these experiments, the EC₅₀ concentration of ME (300 nM), caused an outward current that was approximately 50% of the current induced by ME at a saturating concentration, 30 μM (Figure 7B–a). Labeled LC neurons were also responsive to the α₂-adrenoceptor agonist, UK14304 (3 μM). There was no difference in the amplitude of currents induced by ME between labeled neurons and untreated cells in wild type rats (Figure 7B–b; Osborne and Williams, 1995; Levitt and Williams, 2012; Arttamangkul et al., 2018). Following these experiments, a post hoc analysis of living neurons was evaluated. In each case recorded cell were filled with Alexa 488 during the whole-cell experiment and later imaged using 2-photon microscopy. The A594-labeled MORs were present on the Alexa488-filled neurons. Other nearby LC neurons and dendrites were also labeled with NAI-A594 (Figure 7B–c,d). Thus incubation with a low concentration of NAI-A594 did not affect the concentration response to opioid agonists. When NAI-A594 was used to identify MOR containing neurons in the paraventricular of thalamus areas, all positive staining neurons showed GIRK activation after ME application (n = 4 of 2 slices, data not shown). The results support the advantage of using NAI-A594 to find neurons of interest in mixed populations.

The potential use of NAI-A594 to follow endogenous MOR internalization in brain slices was also studied. NAI-A594 (30 or 100 nM) was incubated in slices of rat LC for 1 hr. Following ME application, the fluorescence of NAI-A594 labeled receptors decreased along the plasma membrane and there were no clear fluorescent puncta in the cytoplasm (Figure 7C–a,a’). To increase the labeling efficiency, LC slices were incubated with high concentrations of NAI-A594 (300 nM or 1 μM) for 2 hr. In this condition, agonist-induced receptor internalization was also not observed. To examine whether the labeling of NAI-A594 might interfere with MOR internalization, LC neurons containing FMOR from transgenic mice were used in the next experiments. Co-labeling with M1-A488 and NAI-A594, revealed fluorescence of both dyes on the plasma membrane (Figure 7C–b,c). After ME application, fluorescent puncta of M1-A488 were found in the cytoplasm and clearly observed in the dendrites (Figure 7C–c,c’). Similar to the results obtained from the rat LC neurons, the fluorescence of NAI-A594 along the plasma membrane decreased without the appearance of fluorescent puncta (Figure 7C–b,b’). The results indicated that while A594-linked receptors were not observed in the endosomes of LC neurons, NAI-A594 labeling did not disrupt the internalization of FMORs labeled with M1-A488.

This study shows that NAI-A594 can be used in low nanomolar concentrations to label endogenous MORs, thus providing the advantage of negligible non-specific background fluorescence and the absence of residual antagonism of NAI-A594 and its product. Incubation times of live brain slices can be varied from minutes to hours at near physiological temperatures. It should be noted that an application of NAI-A594 at a high concentration of 500 nM took only 5 min to stain MORs of LC and PB neurons in the brain slice thus indicating that NAI-A594 can efficiently label endogenous MOR. The weak biological antagonism of NAI-A594 suggests that the compound itself has a low affinity as shown in the binding assays and can be competitively displaced by opioid agonists. Labeling brain slices using low concentrations of NAI-A594 or NAI-A488 can be achieved within an hour. Interestingly, the labeling of purified receptors with NAI-A488 was slower when compared to the labeling of receptors on the living cells. One possible factor is the incubation temperature, which is done at room temperature to stabilize the purified receptors. In addition, the purified receptors may not as frequently form proper conformations for β-naltrexamine to bind and efficiently guide the reaction.

Here we demonstrate that NAI-A594 primarily and covalently labels MOR on neurons in brain slices. This is unexpected given that naltrexamine binds to all three opioid subtypes at low nanomolar concentrations (Li et al., 2009). The design of NAI to have a long linker between the acylimidazole and the amine of naltrexamine (10 atoms apart) was expected to transfer the fluorophore to distant amino acids away from the binding sites. The side chains of Ser, Thr, Tyr or Lys on the second (EL2) or third (EL3) extracellular loops of the receptor are hypothetical sites for modification (Figure 1—figure supplement 1). The ability of fluorescent NAI to predominantly modify MOR likely relies on several factors, including the availability of reactive amino acid side chains and affinity of fluorescent NAI for the different opioid receptors. For example, MOR has 12 amino acids that are potentially capable of reacting with fluorescent NAI, while KOR has 8 and DOR only 3. The paucity of available reactive amino acids in DOR may be one factor contributing to lack of A594 modification in brain slices, despite similar affinities of NAI-A594 for MOR and DOR. Conversely, KOR may have more potential modification sites compared to DOR, but has the lowest affinity for NAI-A594 compared to MOR and DOR (Table 1).

The selectivity of NAI-A594 for MOR over DOR in brain slice experiments contrasts with the experiments in HEK293 cells where the labeling of FMOR and FDOR were comparable. It is possible that the density of DOR on the plasma membrane in vivo is lower compared to MOR (Wang et al., 2008 and references therein). Importantly, the Flag epitope-tagged receptors used in the HEK cells contains a signal peptide to enhance endoplasmic reticulum membrane insertion and plasma membrane expression. The development of compound that is capable of labeling intracellular receptors will be advantage. Changing the fluorescent dye from a hydrophilic and charged Alexa series to one that is smaller and not charged may increase membrane permeability of NAI compounds.

The labeling of MORs with NAI is different from the genetically expressed DOR-GFP and MOR-RFP in knockin mice (Scherrer et al., 2006 and Erbs et al., 2015). Of course the genetically expressed receptors are found both on the plasma membrane and within the cytoplasm in various states of development. Labeling of receptors with NAI-A594 is only plasma membrane associated. The primary advantage of the NAI-A594 is that it is also working with other species particularly rats. The ability to study endogenous receptors in wild type animals of any species offers studies that were otherwise limited to post hoc analysis with antibody labeling.

One key characteristic for a traceless chemical label is that the receptor function is not altered. We confirmed that NAI-A594 was an antagonist at MOR. Pre-application of NAI-A594 (500 nM) decreased receptor activation by nearly half while there was less than 10% inhibition by NAI-A594 at lower concentrations (30–100 nM, data not shown). Like other fluorescent small-molecule ligands, the weak antagonism of NAI-A594 is probably due to the bulkiness of Alexa594 moiety because NAI-AK, the reactive probe without the dye could significantly block MOR activation when used at 300 nM (Figure 7—figure supplement 1). The low antagonism of NAI-A594 is advantageous particularly when only 30–100 nM of NAI-A594 is sufficient for labeling the receptors. In this case, the antagonistic property of NAI-A594 will be minimal and the residual of NAI-A594 or the liberated-naltrexamine product will be removed in a short time. Consistent with this idea, LC neurons labeled with NAI-A594 are able to activate GIRK in response to ME.

In addition to GIRK activation, further evidence that the labeled MOR receptors are functional comes from the agonist-induced internalization studies. Intracellular A594-puncta were observed upon exposure to ME in HEK293 cells expressing FMORs double labeled with M1-488 and NAI-A594 thus indicating that A594-labeled MORs can internalize in response to agonists. Interestingly, when similar internalization experiments are performed on M1-A488/A594 labeled MORs in LC slices, internalized signals appear only from M1-A488. In contrast, there is a noticeable decrease in A594 fluorescence signal on the plasma membrane. A quenching phenomenon of A594 in the acidic environment of endosomes can be ruled out because the fluorescence of A594 is pH insensitive (Panchuk-Voloshina et al., 1999). Surprisingly, similar decline in fluorescence intensities of 30–40% was observed when either ME, morphine or naloxone were applied onto the labeled LC neurons (Figure 3—figure supplement 1A). Because morphine is a non-internalizing agonist and naloxone is an antagonist, the results suggest that the decrease of A594 fluorescence probably occurs on the plasma membrane. This portion of labeling is competitive as also shown in HEK293 cells. Flow cytometry results also showed that a portion of labeling was displaced by naloxone (Figure 2B). The competitive labeling occurs when NAI-A594 occupies the binding site even on receptors that have been previously covalently linked with the fluorescent ligand.

Another potential explanation for the fluorescence decrease may be due to a quenching event caused by nearby amino acids such as tryptophan, tyrosine, histidine and methionine as a result from receptor-conformation change after drug application (Mansoor et al., 2010). However this phenomenon is expected to be reversible after washing out of the drugs but the decrease in fluorescence observed experimentally was not. The decrease in fluorescence intensity after application of drugs might limit visualization of A-594 labeled receptors in the endosomes. In addition, many neurons often show autofluorescence at the excitation wavelengths suitable for A488 and A594, thus further obscuring the detection of low fluorescence of A594 labeled receptors in the endosomes.

The selectivity of NAI-A594 for the MOR makes this reagent a valuable tool to study the functionality and circuitry of MORs in many brain areas. The ‘traceless affinity labeling’ introduced by Hamachi and his colleague is applicable for many membrane proteins including G protein-coupled receptors as shown in this study. The advantage of this ligand-guided labeling is the ability to detect endogenous receptors in living slices. The labeling process is very simple and can be done in any buffer, at room or physiological temperatures and over reasonable time course.

Naloxone and MK-801 were purchased from Hello Bio (Princeton, NJ). [Met⁵]enkephalin and CTAP were purchased from Sigma (St. Louis, MO). UK14304 tartrate and idazoxan were from Tocris (R and D system, Minneapolis, MN). Morphine alkaloid was obtained from National Institute on Drug Abuse, Neuroscience Center (Bethesda, MD) and was converted to HCl salt with 0.1 M HCl as a stock solution. All drugs were diluted to the tested concentrations in artificial cerebrospinal fluid (ACSF) and applied during superfusion. All salts used in electrophysiological experiments were purchased from Sigma.

All animal uses were conducted in accordance with the National Institutes of Health guidelines and with approval from the Institutional Animal Care and Use Committee of OHSU. Animals were housed as a group (2–3 rats or 2–5 mice) to a cage in the animal care facility on a 12-hr light/dark cycle. Food and water were available ad libitum. Juvenile rats (21–28 days) both male and female Sprague-Dawley rats were raised from two breeding pairs that were purchased from Charles River Laboratories (Wilmington, MA). MOR-knockout rats (MOR-KO, Sprague-Dawley) were obtained as breeding pairs from Horizon Discovery (St. Louis, MO) and were bred in house. The Oprm1 gene was inactivated with ZFN target site within exon-2 (Arttamangkul et al., 2018). Both male and female MOR-KO rats were used at the same age as wild type rats. Juvenile mice (21–28 days), both male and female C57BL/6J background were used for all genotypes. Wild type mice were raised from breeders obtained from the Jackson Laboratory (Bar Harbor, ME). MOR-KO mice were gifted from Dr. John Pintar (RWJMS). These mice had Oprm1 gene disrupted in exon-1. Transgenic FLAGMOR mice were generated as described (Arttamangkul et al., 2008). The FLAG epitope was targeted to the N-terminus of murine MOR1 and inserted to the rat tyrosine hydroxylase (Th) gene. Hemizygous animals (FLAGMOR-Tg/+) were used in the experiments. Transgenic THGFP mice were received from Dr. Kobayashi (Sawamoto et al., 2001). The THGFP mice have GFP inserted to the rat tyrosine hydroxylase (Th) gene. The line was maintained as hemizygous.

HEK293 cells stably expressing FLAG epitope-tagged MOR (FMOR, gifted from Dr. Mark von Zastrow, UCSF) and FLAG epitope-tagged DOR (FDOR, gifted from Dr. Manojkumar Puthenveedu, U of Michigan) were grown and maintained in Dulbecco’s minimal essential media (DMEM, Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS), Geneticin sulfate (0.5 mg/ml, ThermoFisher, Waltham, MA) and antibiotic-antimycotic (1x, ThermoFisher). FLAG epitope-tagged KOR (FKOR, the plasmid was obtained from Dr. Mark von Zastrow, UCSF) was transiently transfected into HEK293 cells (ATCC authenticated lines CRL-1573) using Lipofectamine 2000 (ThermoFisher, Waltham, MA). Cell line cultures were free of mycoplasma contamination. Cells were plated onto a cover glass (poly-L-lysine coated, 12 mm, #1 thick, NeuVitro, Vancouver, WA) and used the next day. Cells were incubated in NAI-A594 or NTX-594 (30 nM in cell growth media, 30 min) following with anti-Flag (M1)-A488 (3 μg/ml, 5 min) in the incubator (37°C, 5% CO₂). The cover glass was placed in the imaging chamber and cells were continually perfused with artificial cerebrospinal fluid (ACSF) solution containing (in mM) 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.6 CaCl2, 1.2 NaH2PO4, 11 D-glucose and 21.4 NaHCO3 (95%O2/5%CO2). Experiments to compare labeling of NAI-A594 and NTX-A594 were done at room temperature. Naloxone (1 µM) was superfused for 10 min. Internalization experiments were done at 30°C and with application of [Met⁵]enkephalin (ME, 10 µM) by perfusion for 7–10 min. Imaging was performed as previously described (Birdsong et al., 2013) with an upright spinning disc confocal microscope and a 60x water immersion lens (Olympus LUMIPLAN, NA 1.0). Alexa488 dye and Alexa594 were excited at 488 nm and 561 nm lasers, respectively.

HEK293 cells expressing FMOR were labeled with M1-A594 (3 µg/ml) in buffer A [DMEM containing 5% fetal bovine serum (FBS) and no phenol red] and incubated on ice bath for 1 hr. The conjugated M1-A594 was spectrometrically measured and calculated to have an average of 4 dye molecules per one molecule of IgG. Labeling of cells with NAI-A594 or Natrexamine-A594 (NTX-A594) was done in buffer A and incubated in an incubator at 37°C and 5% CO₂ for 1 hr. All samples were washed three times with DPBS (2 ml, Gibco) and resuspended in ice-cold buffer A (0.3 ml). In some samples, 10 μM naloxone in buffer A was used for washing. Flow cytometry measurements were done using Fortessa (BD Bioscience, Singapore) at the core facility of Oregon Health and Science University (OHSU, Portland, OR). 5000 to 10,000 cells were counted for each condition. Experiments were repeated three times for each condition.

To confirm that NAI-A488 labeling of FMOR expressing cells was specific, lysates were prepared from cells lacking FMOR, from unlabeled FMOR expressing cells, from cells labeled in the presence of naloxone (10 µM), and from cells labeled in the absence of any other treatments. Cell lysates were produced by nutating cells in lysis buffer (20 mM Tris pH 7.4, 100 mM NaCl, 1.0% DDM, 0.5 mM PMSF, 5 ug/ml leupeptin, 1X Halt protease inhibitor cocktail) for 1 hr, at 4°C, followed by centrifugation at 100,000 x g for 30 min and collecting the resulting supernatant. FMOR was immunoprecipitated from cell extracts using a rabbit anti-Mu opioid receptor antibody (Abcam, Cambridge, UK, used at a dilution of 1:100), and 30 µl of a 50% slurry of Protein A Sepharose (GE Healthcare, Marlborough, MA). Approximately 170 µg total protein was immunoprecipitated per each condition. The immunoprecipitated proteins were denatured in sample buffer/0.1 M dithiothreitol (DTT) then split into two aliquots and run on SDS-PAGE, electro-transferred to PVDF membranes, and probed with primary antibodies, either M1 anti-FLAG (Sigma, 1:500 dilution) or rabbit anti-Alexa-488 (ThermoFisher, 1:500 dilution), followed by washing and incubation with secondary antibodies, either goat-anti mouse Alexa-680 (ThermoFisher Scientific, A12058 1:5000 dilution) or goat-anti rabbit Alexa-680 (ThermoFisher Scientific, A12076, 1:5000 dilution). The protein bands were visualized using a LICOR Infrared Imager and Odyssey V3.0 application software. All antibody and wash solutions for blots that were probed with the M1 Mab were supplemented with CaCl₂ (1 mM).

The fluorescence of cell lysates was measured using a fluorimeter (PTI QuantaMaster) with excitation wavelengths (2 nm slit settings) of 470 nm or 590 nm for Alexa-488 or Alexa-594 conjugated compounds, respectively. Fluorescence emission (12 nm slit settings) spectra were collected from 485 to 600 nm (Alexa-488) or from 600 to 700 nm (Alexa-594) using a scan rate of 1 nm/s. The fluorescence emission data were buffer subtracted, normalized to maximal fluorescence in the labeled sample and plotted versus wavelength.

Pellets from FMOR expressing HEK293 cells were solubilized in solubilization buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 30% glycerol, 0.5% n-dodecyl-β-D-maltoside (DDM), 0.3% (3-((3-cholamidopropyl) dimethylammnonio)−1-propanesulfonate (CHAPS), 0.03% cholesteryl hemisuccinate-tris salt (CHS), naloxone (1 μM), Leupeptin (5 μg/ml), PMSF (0.5 mM), 1X Halt protease inhibitor cocktail (ThermoFisher Scientific, 87786, Waltham, MA)], nutated for 1 hr at 4°C, CaCl₂ added to a final concentration of 1 mM, and then centrifuged at 100,000 x g for 1 hr, at 4°C. The resulting supernatant was then applied to an M1 anti-Flag column (Sigma, A4596, St. Louis, MO). 50 μl of M1 bead slurry was used for each ml of clarified lysate. The column was capped and the bead-lysate mixture was then nutated overnight at 4°C. The next morning the column was attached to a ring stand and the beads allowed to settle. The beads were then washed 4X in washing buffer (HEPES, 20 mM pH 7.5, NaCl 150 mM, CaCl₂1 mM, 0.1% DDM, 0.06% CHAPS, 0.006% CHS), followed by elution in washing buffer that lacked CaCl₂ and contained EDTA 2 mM. Eluted FMOR concentrations were determined by UV-Vis spectroscopy (Shimadzu, Kyoto, Japan) using an extinction coefficient, which we estimated from the protein sequence (ExPASy ProtParam tool), for 280 nm absorbance of 62,340 M⁻¹ cm⁻¹. Purity was assessed by SDS-PAGE followed by protein staining with InstantBlue (Expedeon, ISB1L, Sandiago, CA).

Purified FMOR protein was labeled with NAI-A488 at room temperature (RT, 20°C) by incubating FMOR (0.75 μM) with NAI-A488 (4.7 μM) in the absence or presence of naloxone (100 μM) in a final volume of 159 µl. At 0, 4, 24, 48, and 96 hr, 30 µl aliquots were collected and denatured in sample buffer plus 0.1 M DTT. The samples were then run on 10% SDS-PAGE gels. Before incubating gels with InstantBlue protein stain, fluorescent bands were imaged using an Alpha Innotech FluorChem 5500 gel documentation system, using the SYBR green channel. Protein band intensities were determined using Image J.

Rats and mice were anesthetized with isoflurane and killed. The brain was removed and placed in warm (30°C) oxygenated ACSF plus (+)MK-801 (1 µM). Brain slices were prepared (250 µm) using a vibratome (Leica, Nussloch, Germany) and incubated in oxygenated warm (34°C) ACSF containing (+)MK-801 (10 μM) for 30 min before use. Slices were incubated in oxygenated ACSF containing NAI-A594 or NAI-A488 (10–100 nM) for 1 hr at 30°C. Labeled slices were transferred to an upright microscope (Olympus BX51W1, Center Valley, PA) equipped with a custom-built 2-photon apparatus and a 60x water immersion lens (Olympus LUMFI, NA 1.1). The dye was excited at 810 nm for Alexa594 and Alexa568, and 790 nm for Alexa488 and Alexa594 at the same time. Data were acquired and collected using ScanImage Software (Pologruto et al., 2003). Slices were submerged in continuous flow of ACSF at a rate of 1.5 ml/min and drugs were applied via superfusion. All experiments were done at 35°C. Macroscopic images of labeled slices were captured with a Macro Zoom Olympus MVX10 microscope and a MV PLAPO2xC, NA 0.5 lens (Olympus). Alexa594 dye was excited with yellow LED (567 nm). The slices were kept under ACSF in a petri dish during data acquisition. Data were acquired using Q Capture Pro software (Q Imaging, British Columbia, Canada). In the other experiments, a spinning disc confocal microscope and a 20x water immersion lens (Olympus UMPlanFL NA 0.95) was used to take pictures of the labeled brain slices. Alexa488 dye was excited by a 488 nm laser whereas Alexa568 and Alexa594 dyes were excited by 561 nm laser. The slices were maintained alive under superfusion of oxygenated ACSF (30°C).

Whole-cell recordings were done with an Axopatch-1D amplifier in voltage-clamp mode (V_hold = −60 mV). Recording pipettes (1.7–2.1 MΩ, TW150F-3, World Precision Instruments, Saratosa, FL) were filled with internal solution containing (in mM): 115 potassium methanesulfonate or potassium methyl sulfate, 20 KCl, 1.5 MgCl2, 5 HEPES potassium salt, 2 BAPTA, 2 Mg-ATP 0.2 Na-GTP, pH 7.4, 275–280 mOsM. Series resistance was monitored without compensation and accepted for further analysis when < 15 MΩ. Data were collected at 400 Hz with PowerLab (Chart version 5.4.2, AD Instruments, Colorado Springs, CO). [Met⁵]enkephalin was applied by bath superfusion. Bestatin (10 μM, Sigma) and thiorphan (1 μM, Sigma) were included in enkephalin solution to prevent enzymatic degradation. The alpha-2-adrenoceptor agonist, UK14304 (3 µM) and the antagonist Idazoxan (1 µM) were used as controls.

Radioligand binding assays were performed as described (Arttamangkul et al., 1997). Cloned rat mu opioid receptor (MOR) and kappa opioid receptor (KOR), and mouse delta opioid receptor (DOR) stably expressed separately in Chinese hamster ovary (CHO) cells were grown and used to make membrane preparations. [³H]DAMGO, [³H]diprenorphine and [³H]DPDPE were used as radioligands for MOR, KOR and DOR, respectively. Incubations were carried out in triplicate with varying concentrations of NAI-A594 (0.1−10 × 10³ nM) for 90 min at room temperature. DAMGO, Dyn A(1-13)NH₂ and DPDPE were used as control ligands. Nonspecific binding was determined in the presence of 10 µM unlabeled DAMGO, Dyn A(1-13)NH₂, and DPDPE for MOR, KOR and DOR, respectively.

Data were graphed using GraphPad Prism software (La Jolla, CA). All images were processed and analyzed using Image J (NIH, Bethesda, MD).

The synthesis including reagents and chemical characterization of NAI-A594 are described in detail below. β-Naltrexamine was synthesized and purified as described previously (Sayre and Portoghese, 1980).

Chemical structure 1

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Succinic anhydride (1.76 g, 17.6 mmol) was dissolved in 15 mL of anhydrous DMF, under nitrogen atmosphere. A solution of histamine −2 HCl (2.93 g, 15.9 mmol) in 25 mL anhydrous DMF and triethylamine (31.8 mmol, 4.43 mL) was added dropwise and stirred. The reaction mixture was carried on overnight at room temperature. The precipitate was filtered and the filtrate was concentrated under vacuum. Ethanol (20 mL) was added to the concentrated solution and the mixture was chilled at 4°C overnight. The white precipitate was filtered, washed with cold ethanol, and dried in vacuo. 2.83 g of a white solid was obtained (84%). 1H NMR (D₂O): δ (ppm) 8.5 (s, 1H), 7.20 (s, 1H), 3.41 (t, J = 6.37 Hz, 2H), 2.86 (t, J = 6.37 Hz, 2H), 2.35 (s, 4H).

Chemical structure 2

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Naltrexamine 2HCl (9.97 mg, 24 µmol) was dissolved in DMF to give 16 mM solution. The naltrexamine solution (1.5 mL) was transferred to a 2.5 mL microwave vial, following with compound 1 (20.3 mg, 96 µmole), HBTU (72 µmole, 27.3 mg), triethylamine (two drop,~40 uL). Additional of 1 mL DMF was added to wash down the reagents on the side of vial. Microwave reactor was set to 150° C, 10 min, normal absorption. HPLC analysis of the crude material using a gradient solution of 5% to 65% water and acetonitrile containing 0.1% trifluoroacetic acid in 30 min and a reverse phase (Jupiter, 5 µm, C18, 300 Å, 250 × 4.6 mm) confirmed the reaction was completed and there was no naltrexamine in the crude. The crude was purified using silica gel chromatography (DCM/MeOH/NH4OH 4/1/1). The eluent was collected and evaporated. The electron-spray ionized mass spectrum confirmed the product peak at 536.5 (m/z+1). Yields 12 mg (94%). 1HNMR (400 MHz, CD₃OD): δ (ppm) 8.76 (s, 1H), 7.36 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 4.68 (d, J = 8 Hz, 1H), 4.37 (m, 2H), 4.22 (s, 2H), 3.98 (m, 1H), 3.80 (t, J = 4 Hz, 2H), 3.61 (m, 1H), 3.14 (m, 1H), 2.92 (m, 3H), 2.66 (m, 3H), 2.55 (m, 2H), 2.44 (m, 2 hr), 1.90 (m, 1H), 1.67 (m, 3H), 1.12 (m, 1H), 0.85 (m, 1H), 0.76 (m, 1H), 0.53 (m, 2H). MS (ESI pos) m/z found 536.5 (M + H).

Chemical structure 3

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A solution of 2-(Prop-2-yn-1-yloxy)ethanol (25.0 mg, 0.25 mmol), N,N’-disuccinimidyl carbonate (96 mg, 0.375 mmol), and triethylamine (140 uL, 1 mmol) in CH₃CN (3 mL) was stirred for 2 hr at 40°C. The solvent was evaporated and product was purified using silica gel chromatography (0% to 50% ethyl acetate/hexane). The product was detected by vanillin stain. The eluent was collected and evaporated giving the product of clear oil. Yielded 48 mg (80%). 1HNMR (400 MHz, CDCl₃): δ (ppm) 4.56 (m, 2H), 4.19 (d, J = 2.38 Hz, 2H), 3.80 (m, 2H), 2.81 (s, 4H), 2.52 (t, J = 2.38 Hz, 1 hr).

Chemical structure 4

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A solution of compound 3 (48 mg, 200 µmol) in 2 mL DMF was combined with compound 2 (~24 µmol) in a vial. Pyridine five drops were added, and the reaction mixture was stirred at room temperature overnight. Then the reaction mixture was filtered over celite. The filtrate was concentrated and azeotroped with toluene. The residue was washed twice with ether. Ethyl acetate was added to give a solid product. Filtrate of excess compound 3 was removed. The solid product was dissolved in methanol and filtered over celite. Methanol was then evalprated under vacuo and light brown solid powder was obtained. Yields 4.5 mg (66%). The electron-spray ionized mass spectrum confirmed the product peak at 662.6 (m/z+1). 1HNMR (400 MHz, CD3OD): δ (ppm) 8.22 (s, 1H), 7.40 (s, 1H), 6.74 (d, J = 2.12 Hz, 1H), 4.57 (m, 3H), 4.24 (d, J = 2.4 Hz, 2H), 3.90 (m, 3H), 3.63 (m, 1H), 3.46 (m, 2H), 3.16 (m, 2H), 2.87 (m, 3H), 2.76 (t, J = 6.83 Hz), 2.70 (m, 2H), 2.49 (m, 4H), 1.87 (m, 1H), 1.66 (m, 4 hr), 1.34 (m, 1 hr), 1.22 (m, 1H), 0.84 (m, 1H), 0.76 (m, 1H). MS (ESI pos) m/z found 662.1 (M + H).

Compound 4 (0.75 mg, 2.27 mM) in water was added to a solution of AFdye 594 azide (Click Chemistry Tool, Scottsdale, AZ) in methanol (1 mg, 1.6 mM). A freshly-made ascorbate solution (150 µl, 100 mM, adjust pH to 4.0) was added to the mixture and stirred at room temperature. To this solution, a mixture of CuSO₄ (12 µl, 25 mM) and BTTES (18 µl, 50 mM) was added and stirring was continued for 2.5 hr. HPLC analysis of the crude material using a gradient solution of 5% to 65% water and acetonitrile containing 0.1% trifluoroacetic acid in 30 min and a reverse phase (Jupiter, 5 µm, C18, 300 Å, 250 × 4.6 mm) showed two major peaks. The peak at 25 min was identified as the product and at 28 min as the free dye by dual wavelength UV-detection (at 220 and 594 nm). The crude material was then purified using a preparative HPLC system using the same gradient condition and a preparative reverse phase column (Jupiter, 5 µm, C18, 300 Å, 250 × 21.2 mm). The purity of product was greater than 95% by HPLC analysis. The collected fractions were combined and lyophilized to obtaind dried powders. The electron-spray ionized mass spectrum confirmed the product peak at 755.2 (m/z +2). The product was dissolved in methanol and measured OD at maximal OD of 591 nm. Using absorption extinction (88,000) of free dye provided by the company (Click Chemistry Tools), the yield was 0.81 mg or 47%. NAI-A488 was prepared in a similar method that yielded 49% product.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

The authors thank Drs. Maria Torecilla and Brooks Robinson for their comments and technical help with brain slice preparations. We also thank the Medicinal Chemistry Core (Dr. Aeron Nilson), Flow Cytometry Core (Pamela Canaday) and Bioanalytical Shared Resource/Pharmacokinetic Core (Jenny Luo) at OHSU for their superior services. The work is funded by grants DA008163-JTW, DA048136-JTW, DLF and SA, and DA042779-WTB from the National Institutes of Health. A portion of this work was supported the Intramural Research Programs of the National Institute on Drug Abuse and National Institute of Alcohol Abuse and Alcoholism.

Animal experimentation: All animal uses were conducted in accordance with the National Institutes of Health guidelines and with approval from the Institutional Animal Care and Use Committee (IACUC) protocol #IP00000160 of the Oregon Health & Science University. Rats and mice were anesthetized with isofluorane before euthanized with minimal suffering.

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