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Opioids, palatable feeding and exercise

Ugur, M.

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Chapter 4

Morphine-dependent and abstinent mice

are characterized by a broader

distribution of the neurons co-expressing

mu and delta opioid receptors

M Ugur*, F Pierre*, F Faivre, S Doridot,

P Veinante, D Massotte

Neuropharmacology. In press 2019

96

CHAPTER 4

Morphine-Dependent and Abstinent Mice are characterized by a

Broader Distribution of the Neurons co-expressing Mu and Delta

Opioid Receptors

Florian Pierre*, Muzeyyen Ugur*, Fanny Faivre, Stéphane Doridot, Pierre Veinante,

Dominique Massotte

Centre de la Recherche Nationale Scientifique, Université de Strasbourg, Institut des

Neurosciences Cellulaires et Intégratives, Strasbourg, France

* these authors contributed equally to the work

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ABSTRACT Opiate addiction develops as a chronic relapsing disorder upon drug recreational use or following

misuse of analgesic prescription. Mu opioid (MOP) receptors are the primary molecular target of

opiates but increasing evidence support in vivo functional heteromerization with the delta opioid

(DOP) receptor, which may be part of the neurobiological processes underlying opiate addiction. Here, we

used double knock-in mice co-expressing fluorescent versions of the MOP and DOP receptors to examine

the impact of chronic morphine administration on the distribution of neurons co-expressing the two

receptors. Our data show that MOP/DOP neuronal co-expression is broader in morphine- dependent

mice and is detected in novel brain areas located in circuits related to drug reward, motor activity,

visceral control and emotional processing underlying withdrawal. After four weeks of abstinence,

MOP/DOP neuronal co-expression is still detectable in a large number of these brain areas except in the

motor circuit. Importantly, chronic morphine administration increased the proportion of MOP/DOP

neurons in the brainstem of morphine-dependent and abstinent mice. These findings establish

persistent changes in the abstinent state that may modulate relapse and opiate-induced hyperalgesia

and also point to the therapeutic potential of MOP/DOP targeting.

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List of abbreviations 12N: hypoglossal nucleus; AA: anterior amygdaloid area; ACo: anterior cortical amygdaloid

nucleus; AHC: anterior hypothalamic area, central part; AHP: anterior hypothalamic area,

posterior part; AI: agranular insular cortex; Amb: ambiguus nucleus; APT: anterior pretectal

nucleus; B: basal nucleus; Bar: Barrington's nucleus; BSTIA: bed nucleus of the stria terminalis,

intraamygdaloid division; BSTLV: bed nucleus of the stria terminalis, lateral division, ventral part;

CA1: field CA1 of hippocampus; CA3: field CA3 of hippocampus; CeI: central amygdaloid

nucleus,intermediate part; CPu, dl: caudate putamen, dorsolateral; DA11: DA11 dopamine

cells; DEn: dorsal endopiriform nucleus; DG: dentate gyrus; DOP: delta opioid receptor; DRC:

dorsal raphe nucleus, caudal part; DRD: dorsal raphe nucleus, dorsal part; DTgC: dorsal tegmental

nucleus, central part; DTT: dorsal tenia tecta; Ecu: external cuneate nucleus; GiA: gigantocellular

reticular nucleus, alpha part; GiV: gigantocellular reticular nucleus, ventral part; IC: inferior

colliculus; IF5: interfascicular trigeminal nucleus; ILL: intermediate nucleus of the lateral

lemniscus; InG: intermediate grey layer of the superior colliculus; IPAC: Interstitial nucleus of the

posterior limb of the anterior commissure; IRt: intermediate reticular nucleus; IS: inferior

salivatory nucleus; KF: Kölliker-Fuse nucleus; Lat: lateral (dentate) cerebellar nucleus; LC: locus

coeruleus; LGP: lateral globus pallidus; LH: lateral hypothalamic area; LPAG: lateral

periaqueductal gray; LPB: lateral parabrachial nucleus; LPGi: lateral paragigantocellular nucleus;

LRt: lateral reticular nucleus; LSD: lateral septal nucleus, dorsal part; LSI: lateral septal nucleus,

intermediate part; LVPO: lateroventral periolivary nucleus; MCLH: magnocellular nucleus of the

lateral hypothalamus; MdD: medullary reticular nucleus, dorsal part; Me5: mesencephalic

trigeminal nucleus; MeA: medial amygdaloid nucleus; Med: medial (fastigial) cerebellar nucleus;

MePD: medial amygdaloid nucleus, posterodorsal part; MePV: medial amygdaloid nucleus,

posteroventral part; MOP: mu opioid receptor; MPB: medial parabrachial nucleus; mRt:

mesencephalic reticular formation; MVPO: medioventral periolivary nucleus; Pa4: parathrochlear

nucleus; PaF: parafascicular thalamic nucleus; PCRtA: parvicellular reticular nucleus, alpha

part; PeF: perifornical nucleus; Pir: piriform cortex; PMnR: paramedian raphe nucleus; Pn:

pontine nucleus; PnC: pontine reticular nucleus,caudal part; PnO: pontine reticular nucleus,oral

part; Pr: prepositus nucleus; Pr5: principal sensory trigeminal nucleus; PSTh: parasubthalamic

nucleus; PTe: paraterete nucleus; PTg: pedunculotegmental nucleus; RIP: Raphe interpositus

nucleus; RLi: rostral linear nucleus of the raphe; RM: retromammillary nucleus; RMC: red

nucleus, magnocellular part; RMg: raphe magnus; RPa: raphe pallidus; RPC: red nucleus,

parvicellular part; RR: retrorubral nucleus; RtTg: reticulotegmental nucleus of the pons;

RVL: rostroventrolateral reticular nucleus; RVM: rostral ventromedial medulla; S: subiculum;

SHi: septohippocampal nucleus; SNC: substantia nigra, compact part; SNR: substantia nigra,

reticular part; Sol: nucleus of the solitary tract; Sp5: spinal trigeminal nucleus; STh: subthalamic

nucleus; SubB: subbrachial nucleus; Tu: olfactory tubercle; Tz: nucleus of the trapezoid body;

VCN: ventral cochlear nucleus; Ve: vestibular nuclei; VLL: ventral nucleus of the lateral lemniscus;

VP: ventral pallidum; VTA: ventral tegmental area; ZI: zona incerta

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INTRODUCTION The opioid system modulates numerous physiological functions such as nociception,

emotional responses, reward and motivation, but also controls the autonomic nervous system. At

the molecular level, mu opioid (MOP) receptors constitute the primary target of opiates and

mediate their analgesic and euphoric properties. However, several decades of pharmacology have

uncovered the complexity of the opioid system. More specifically, analysis of opiates effects in vivo

revealed functional cross-talk between MOP and another opioid receptor subtype, the delta

opioid (DOP) receptor, which may be involved in the development of tolerance (1).

Numerous reports indicate that co-expression of MOP and DOP receptors in heterologous

systems affects their binding and signaling properties through MOP/DOP heteromer formation

(2) and data accumulate to support the presence of functional MOP/DOP heteromers in vivo.

Evidence of close physical proximity at the supraspinal level included detection with MOP/DOP

selective antibodies (3), MOP/DOP receptor co-immunoprecipitation in the hippocampus, (4) and

antinociceptive activity upon intracerebroventricular (i.c.v.) injection of MDAN-21, a bivalent

ligand bridging the MOP agonist oxymorphone and the DOP antagonist naltrindole (5). Direct

physical interaction was established in the nucleus accumbens, spinal cord and dorsal root

ganglia through disruption of co- immunoprecipitation by an interfering peptide respectively

corresponding to the DOP carboxy tail (6), the MOP transmembrane domain TM1 (7) or the DOP

second intracellular loop IL2 (8).

On the functional point of view, co-activation of MOP and DOP receptors in native tissue resulted

in a positive crosstalk that can contribute to increase neuronal hyperpolarization (9, 10).

MOP/DOP heteromers showed preferential coupling to the pertussis toxin insensitive Gαz subunit

that would not be desensitized by chronic morphine administration in the rat striatum and

hippocampus (11, 12). Activation of MOP/DOP heteromers also increased β-arrestin signaling

(13). Although disruption of the physical contact between MOP and DOP receptors was reported to

facilitate morphine analgesia (7) and to reduce morphine tolerance (7, 8), MOP/DOP preferential

activation by the agonist CYM51010 or the bivalent ligand MDAN-21 produced acute thermal

analgesia comparable to morphine but induced less tolerance and physical dependence upon

repeated administration (5, 14). This designates MOP/DOP heteromers as potential novel

therapeutic targets in the context of chronic pain and opiate addiction. Importantly, chronic

morphine administration increased DOP receptor localization at the cell surface in the central

and peripheral nervous system through a MOP receptor dependent mechanism (1, 15, 16).

In addition, ELISA using a MOP/DOP specific antibody detected enhanced MOP/DOP density

in brain membranes from mice chronically treated with morphine (3).

We previously mapped neurons co-expressing MOP and DOP receptors in the brain using

double fluorescent knock-in mice co-expressing a green fluorescent version of DOP receptors (DOP-

eGFP) and a red fluorescent version of MOP receptors (MOP-mCherry). This study revealed

MOP/DOP neuronal co-localization in discrete neuronal populations located in subcortical

networks essential for survival, including the perception and processing of aversive stimuli (4).

MOP/DOP co-expression was especially abundant in brainstem nuclei tightly connected with the

autonomic nervous system where they may functionally cooperate to produce somatic and

autonomic symptoms during withdrawal (4). Here, we mapped changes in MOP/DOP

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distribution throughout the brain after chronic morphine administration in order to get better

insight into the neurobiological processes underlying chronic distribution throughout the

brain after chronic morphine administration in order to get better insight into the

neurobiological processes underlying chronic opiate administration. We also charted

alterations in MOP/DOP neuronal co-expression after four weeks abstinence to determined

long-lasting changes induced by chronic morphine administration.

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MATERIALS AND METHODS

Animals Double knock-in mice co-expressing fluorescent MOP and DOP receptors (MOP-mCherry / DOP-

eGFP) were obtained by crossing previously generated single fluorescent knock-in mice expressing

DOP-eGFP or MOP-mCherry, as described previously (4). The genetic background of all

animals was 50:50 C57BL6/J:129svPas. Male and female adult mice (8 to 12 weeks old) were used.

Mice were housed in an animal facility under controlled temperature (21 ± 2 °C) and humidity

(45 ± 5 %) under a 12:12 dark–light cycle with food and water ad libitum. All experiments were

performed in agreement with the European legislation (directive 2010/63/EU acting on

protection of laboratory animals) and received agreement from the French ministry (APAFIS

20 1503041113547 (APAFIS#300.02).

Animal treatment Physical dependence to morphine was verified in a parallel group of mice (n=7 saline, n=7

morphine). Experiments were performed in stable conditions: 21 ± 2 °C, 45 ± 5 % humidity,

40 ± 2 lux. All experiments were preceded by 2 days of animal handling. Mice daily received

intraperitoneal injection (i.p.) of 30 mg/kg morphine or of a saline solution for 6 days. Physical

dependence to morphine was verified by measuring withdrawal syndrome precipitated by a

naloxone (1mg/kg, i.p.) injection 2 hours after the last morphine injection. Somatic and vegetative

signs of withdrawal were scored by period bins of 5 minutes for 20 minutes. Horizontal activity

(scored from 0 to 2) and rearing (expressed as event numbers) were measured to characterize

global activity. Scratches, genital licks, grooming, head shakes, wet dog shakes, paw tremors,

sniffing (expressed as event numbers) and body tremors (absence or presence scored as 0

or 1) were measured as anxiety and discomfort signs. Finally, vegetative signs such as ptosis,

piloerection, teeth chattering and diarrhea were observed (absence or presence scored as 0 or 1

respectively). We established a global score of withdrawal that took into account the relative

weight of each scored sign and calculated this score for each mouse as previously described (17).

A global withdrawal score was calculated as follows (activity + rearing + grooming + genital licks

+ scratches + wet dog shakes) x 0.5 + (jumps + paw tremors + sniffing + body tremors + ptosis +

piloerection + teeth chattering + diarrhea) x 1. Following chronic morphine administration,

abstinent animals were housed for 4 weeks in their home cages.

Tissue preparation and immunohistochemistry Animals were injected with the DOP selective agonist SNC80 1 hour before perfusion to

facilitate detection of the soma of DOP-eGFP neurons according to Erbs et al. 2015 (4). Tissue

preparation and amplification of the eGFP signal by immunohistochemistry were performed as

previously described (4). Briefly, mice were anaesthetized with ketamine/xylazine (100/10

mg/kg, i.p.) and perfused intracardiacally with 100 ml 4% paraformaldehyde (PFA) (at 2-4°C)

in PB 0.1M pH7.4 at 20ml/min. Brains were post-fixed for 24 hours at 4°C in 4% PFA solution,

cryoprotected at 4°C in a 30 % sucrose, PB 0.1M pH 7.4 solution, embedded in OCT (Optimal Cutting

Temperature medium, Thermo Scientific), frozen and kept at -80°C. Floating 30 µm thick brain

sections were cut with a cryostat (CM3050, Leica) and incubated in blocking solution (PB 0.1M pH

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7.4, 0.5% Triton X100 (Sigma, St Louis, MO, USA), 5% normal goat serum (Invitrogen, Paisley,

UK)) for 1 hour at room temperature (RT). Sections were incubated overnight at 4°C in the

blocking solution with a rabbit anti-GFP antibody (Molecular Probes A-6455, dilution 1:1000)

antibody. Sections were washed three times with PB 0.1M pH 7.4, 0.5% Triton X100, incubated for

2 hours at RT with AlexaFluor 488 conjugated secondary antibodies (Molecular Probes A-11034,

dilution 1:2000). For neuronal co-localization, mouse monoclonal antibodies raised against

parvalbumin (Swant cat Nr 235, dilution 1:1000), calbindin D-28K (Swant cat Nr 300, dilution

1:1000) or rat monoclonal antibodies raised against somatostatin (Millipore MAB 354, dilution

1:100) were detected with AlexaFluor 647 conjugated goat anti-mouse (Molecular Probes A-

21240, dilution 1:500) or Dylight 650 conjugated goat anti-rat (Invitrogen SA5-10021, dilution

1:500) antibodies. Sections were washed three times and mounted on SuperfrostTM glass

(Menzel-Glaser) with Mowiol (Calbiochem, Darmstadt, Germany) and 4', 6-diamidino-2-

phenylindole (DAPI) (Roche Diagnostic, Mannheim, Germany) (0.5 µg/ml).

Image acquisition and analysis

Image acquisition was performed with the slide scanner NanoZoomer S60 and fluorescence

module L11600-21 (Hamamatsu Photonics, Japan). The light source LX2000 (Hamamatsu

Photonics, Japan) consisted in an ultra-high-pressure mercury lamp coupled to an optical fiber.

Single RGB acquisition was made in the epifluorescence mode with a filter-set optimized for

DAPI, fluorescein, tetramethylrhodamine or Cy5 detection. The acquisition was performed using

a dry 20x objective (NA: 0.75). The 40x resolution was achieved with a lens converter. The latter

mode used the full capacity of the camera (resolution: 0.23 µm/pixel). Neurons expressing a given

fluorescent marker are visualized using the NDP viewer system with an integrated high-resolution

zoom and possibility to separate the different fluorescent components.

Observations with a confocal microscope (SP5RS, Leica) using 40x (NA: 1.25) and 63x (NA: 1.4)

oil objectives were used to validate MOP and DOP receptor co-localization. Images were acquired

with the LCS (Leica) software. Confocal acquisitions were performed in the sequential mode

(single excitation beams: 405, 488 and 568 nm) to avoid potential crosstalk between the

different fluorescence emissions.

Brain regions were identified using the 4th edition of the Mouse Brain Atlas Paxinos and Franklin

(18). Counting was performed on non-consecutive sections in the dentate gyrus (DG), the

Ammon’s horn 3 (CA3) and the Ammon’s horn 1 (CA1) regions of the dorsal hippocampus

(Bregma: -1.58 mm to -1.94 mm), in the locus coeruleus/Barrington nucleus (Bregma: -5.33 mm

to -5.63mm) and in the rostral ventromedial medulla (Bregma -5.5 to -6.5) on areas including the

posterior raphe nuclei (pallidus, magnus, obscurus, interpositus), the parapyramidal nucleus,

the alpha part of the gigantocellular reticular nucleus and lateral paragigantocellular nucleus.

Statistical analysis Statistical analysis was performed with Graph-Pad Prism v7 (GraphPad, San Diego, CA).

Changes between saline, chronic morphine and morphine abstinent animals was analyzed

in each area independently using one-way ANOVA with multiple comparisons followed by

Dunnet’s test for post- hoc analysis. All data were normally distributed as first verified using the

Shapiro-Wilk normality test. Student’s t test was used for behavioral analysis.

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RESULTS Chronic morphine administration induces physical dependence We first verified that chronic morphine treatment elicited physical dependence under our

conditions. A group of mice chronically treated with daily injection of morphine (30mg/kg i.p.)

was subjected to naloxone-precipitated withdrawal. Somatic and vegetative signs (horizontal

activity, paw and body tremors, head shakes and wet dog shakes, jumps, ptosis, teeth chattering,

piloerection and diarrhea) were scored. As expected, morphine-treated mice exhibited higher

global withdrawal score compared to saline treated animals (unpaired Student’s t test t(12)=7.689,

p<0.0001) (Fig. 1). Chronic morphine- treated animals therefore exhibited physical

dependence.

Figure 1. Chronic morphine treatment induces a drug-dependent state in mice. Global score of pharmacological

withdrawal precipitated by naloxone (1mg/kg i.p.) in mice daily treated with morphine 30 mg/kg (i.p.) or in saline-

treated controls. n=7 per group. Student’s t test, ****p<0.0001

Chronic morphine alters MOP/DOP neuronal distribution In morphine-dependent mice, neurons co-expressing MOP-mCherry and DOP-eGFP were

identified in 85 brain regions (Table 1, Figs. 2, 3 and 4). About half of these structures (44) have

been previously described in control saline animals (4) (Table 1, Figs. 2A, 3 and 4). These

regions include (1) a few forebrain areas such as the hippocampus, the piriform cortex, the lateral

globus pallidus and the basal nucleus as well as parts of the hypothalamus (anterior, lateral,

retromammillary), (2) midbrain structures (red nucleus, substantia nigra pars reticulata, ventral

tegmental area), and (3) numerous hindbrain regions, essentially involved in modulatory

processes (raphe nuclei, locus coeruleus) or in somatic and visceral sensorimotor functions

(reticular formation, trigeminal, auditory and vestibular nuclei).

In addition to these regions, we identified 41 new brain structures where neurons co-expressing

MOP- mCherry and DOP-eGFP were only detectable after chronic morphine treatment (Table 1,

Figs. 2B and 4). Half of them were found in the forebrain, mainly in the basal ganglia, basal forebrain,

amygdala and extended amygdala, and hypothalamus. The other half was located in the

brainstem, including the reticular areas, auditory regions, parabrachial area, raphe nuclei and

visceral nuclei. Our results thus establish wider distribution of MOP/DOP neurons which is

not restricted to the hindbrain but also expands to forebrain areas

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Table 1. Distribution of MOP/DOP neurons in the brain of saline, chronic morphine and abstinent mice

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Figure 2. Brain mapping of MOP/DOP co-expressing neurons. Distribution of neurons co-expressing MOP-mCherry and

DOP- eGFP in (A) brain areas common to saline, morphine-dependent and abstinent mice. (B) New areas of MOP-

mCherry and DOP-eGFP neuronal co-expression following chronic morphine administration that are no longer detected

after four weeks abstinence (blue circle) or are still present in abstinent mice (red circle). n=4 saline, n=3 chronic

morphine, n=4 abstinent mice.

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Figure 3. MOP/DOP neuronal co-expression in the hippocampus of saline, morphine-dependent and abstinent

mice. General view of the regions (top panel). Scale bars 500 µm. Representative images showing MOP-mCherry and DOP-

eGFP co- expression in the same neurons in the CA1, CA3 and DG. MOP/DOP neurons are indicated by arrows. Scale

bars 100 µm.

Figure 4. MOP/DOP neuronal co-expression in various brain regions of saline, morphine-dependent and abstinent

mice. General view of the regions (top panel). Scale bars 500 m. Representative images showing MOP-mCherry and

DOP-eGFP expression in the different areas. Scale bars 100 m Inset: enlargement showing MOP/DOP neuronal co-

expression.

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Changes in MOP/DOP neuronal distribution persist in abstinent mice In abstinent mice, no additional areas containing neurons co-expressing MOP-mCherry and DOP-

eGFP were identified compared to saline and/or morphine dependent animals. Indeed, we

observed MOP/DOP neurons in 74 brain structures, among which the 44 identified in saline

animals (Table 1, Figs. 2A, 3 and 4) and 30 structures identified in morphine-treated animals

(Table 1, Figs. 2B and 4). Notably, MOP/DOP co-localization induced by chronic morphine

administration persisted in the forebrain of abstinent mice in the insular cortex, olfactory tubercle,

dorsolateral caudate putamen, the limit between dorsal and intermediate lateral septum, ventral

pallidum, amygdala (anterior area, anterior cortical nucleus), and extended amygdala (central and

medial nuclei). Similarly, double labeled neurons were still present four weeks after chronic

morphine treatment in the midbrain (inferior colliculus, magnocellular red nucleus, substantia

nigra pars compacta, medial terminal nucleus, dopaminergic group DA11, mesencephalic

reticular formation, mesencephalic trigeminal nucleus), the pons (nuclei of the lateral lemniscus,

dorsal raphe, medial parabrachial nucleus and Kölliker-Fuse nucleus) and in the medulla (raphe

interpositus nucleus, lateral reticular nucleus, inferior salivatory nucleus and solitary nucleus).

Altogether, these data indicate that chronic morphine administration durably affects

MOP/DOP neuronal distribution with about 70% of the areas newly identified in morphine

dependent mice still detectable after four weeks of abstinence.

Impact of chronic morphine treatment on MOP/DOP neurons in the hippocampus We then examined in more detail the impact of chronic morphine administration on

MOP/DOP neurons in the hippocampus where the existence of functional MOP/DOP heteromers

is established (4, 19).

Using DOP-eGFP knock-in mice, we previously showed that DOP-eGFP neurons are GABAergic

with 70% of the DOP-eGFP neurons co-expressing parvalbumin, 16% co-expressing somatostatin

and 14% co-expressing calbindin in basal conditions (20). Here, we observed a similar distribution for

MOP/DOP positive neurons with 70% of the MOP/DOP neurons co-expressing parvalbumin, 17%

co-expressing somatostatin and 12% co-expressing calbindin (Fig. 5).

We then investigated the impact of chronic morphine administration on the different

populations expressing the MOP and/or DOP receptors. In agreement with our previously

published data (16), the density of DOP-eGFP neurons was decreased in morphine treated animals

compared to saline controls in the CA1 (80 ± 6%), CA3 (69 ± 9%) and dentate gyrus (DG) (53±

14%) (Fig. 6A, Table 2). Similarly, chronic morphine administration decreased the density of MOP-

mCherry neurons compared to saline controls in the CA1 (88 ± 8%), the CA3 (83 ± 11%) and the DG

(63 ± 8%) (Fig 6B, Table 2). Accordingly, the density of MOP/DOP neurons also decreased in the

CA1 (85 ± 7 %), the CA3 (78 ± 9 %) and in the DG (36 ± 10%) following chronic morphine

administration (Fig. 6C, Table 2).

Importantly, chronic morphine administration did not modify the proportion of DOP or MOP

neurons co-expressing the other receptor compared to saline control mice (Fig. 6D,E, Table 2).

Accordingly, the proportion of MOP/DOP neurons was not affected (Fig. 6F, Table 2). Indeed,

MOP/DOP neurons represented 21.2 ± 1.3% of the total population expressing MOP or DOP

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receptors in the CA1 compared to 20.2 ±0.5 % in saline controls. In the CA3, they constituted

18.6 ± 1.5 % versus 18.7 ± 0.9% in saline controls and, in the DG, they represented 11.2 ± 2.6%

versus 11.5 ± 2.2% in saline controls (Fig. 6F, Table 2).

Table 2. Statistical analysis related to neuronal MOP/DOP co-expression

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Table 2. continued

Figure 5. MOP/DOP neurons are GABAergic in the hippocampus. General view of the hippocampus showing co-

localization of MOP-mCherry and DOP-eGFP with (A) parvalbumin, (B) somatostatin, and (C) calbindin markers. (D)

Neuronal co-localization of MOP-mCherry, DOP-eGFP and parvalbumin, (E) neuronal co-localization of MOP-mCherry,

DOP- eGFP and somatostatin, (F) neuronal co-localization of MOP-mCherry, DOP-eGFP and calbindin. Scale bars

10 µm.

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Figure 6. Impact of chronic morphine administration on DOP, MOP and MOP/DOP populations in the hippocampus.

Density of (A) DOP, (B) MOP and (C) MOP/DOP neurons was decreased in the CA1, CA3 and dentate gyrus (DG) of

chronic morphine treated (light grey bars) and abstinent (dark grey bars) mice compared to saline controls (white bars).

The relative proportion of (E) DOP, (F) MOP, and (G) MOP/DOP neurons remained unaffected in the CA1, CA3 and DG

of chronic morphine treated (light grey bars) and abstinent (dark grey bars) mice compared to saline controls (white

bars). n=6 saline, n=5 chronic morphine, n=5 abstinent mice. One-way ANOVA, Dunnett’s post-hoc analysis,

*p<0.05, **p<0.01, ***p<0.001.

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We then examined whether four weeks of abstinence reversed the effects of chronic

administration of morphine. As previously observed for the DOP-eGFP distribution (16), the

neuronal density of DOP expressing neurons remained lower compared to saline control (70 ±

13% in the CA1, 77 ± 9% in the CA3 and 48 ± 10 % in the DG) (Fig 6A, Table 2). Decreased density

was still detected in the CA1 (65 ± 4%) and in the DG (70 ± 14%) but not in the CA3 (80 ± 9%) for MOP

expressing neurons (Fig 6B, Table 2). Similarly, the density of MOP/DOP expressing neurons

remained lower in the CA1 (74 ± 10 %) and DG (47 ± 10 % ) but was comparable to control in the CA3

( 94 ± 15%) (Fig. 6C). Again, no change was observed in the proportion of DOP (Fig. 6D) or MOP (Fig.

6E, Table 2) neurons co-expressing the other receptor or in the proportion of MOP/DOP neurons

within the total population (23.2 ± 1.4% in the CA1, 21.2 ± 2.6 % in the CA3 and 12.2 ± 2.1% in

the DG) (Fig. 6F, Table 2).

Altogether, our data indicate that chronic morphine administration durably decreased the density

of MOP/DOP neurons in the hippocampus but did not modify its relative proportion within the

total population of neurons expressing MOP or DOP receptors.

Impact of chronic morphine treatment on MOP/DOP neurons in the hindbrain We then investigated the impact of chronic morphine in the hindbrain where MOP/DOP neuronal

co- localization is abundant. We analyzed changes in the locus coeruleus/Barrington (LC/Bar) nuclei

and in the rostral ventromedial medulla (RVM) that are both tightly connected to the autonomic

nervous system.

In the LC/Bar nuclei, chronic morphine increased the density of DOP neurons (186 ± 35 %) (Fig.

7A, Table 2), MOP neurons (150 ± 39 %) (Fig. 7B, Table 2) and MOP/DOP neurons (309 ± 99%)

(Fig. 7C, Table 2) compared to saline controls. The proportion of DOP positive neurons increased

compared to the saline condition (71 ± 10% vs 35 ± 7 % respectively) (Fig. 7D Table 2). A similar trend

was observed for the proportion of MOP positive neurons also expressing DOP receptors (38± 13 %

vs 15 ± 5 %) (Fig. 7E, Table 2) and the proportion of MOP/DOP neurons (24 ± 7 % vs 9 ± 3%) (Fig 7F,

Table 2). In abstinent conditions, the density of DOP (97 ± 19%), MOP (69 ± 7%) and DOP-MOP

(175 ± 50%) neurons were decreased compared to morphine-dependent animals and not

statistically different from the saline condition (Fig. 7 A-C, Table 2). However, the proportion of DOP

positive neurons co-expressing MOP receptors (74 ± 9 %) (Fig. 7D, Table 2) and of MOP positive

neurons also co-expressing DOP receptors (34 ± 7 %) remained similar to what was observed in

morphine treated mice and higher than in the saline control condition (Fig.E, Table 2).

Accordingly, a statistically significant increase in MOP/DOP neurons was detected compared to

the saline condition (25 ± 5 % vs 9 ± 3 %) (Fig. 7F, Table 2)

In the RVM, chronic morphine administration did not significantly affect the density of DOP

neurons (124 ± 15 %), MOP neurons (99 ± 10%) or MOP/DOP neurons (170 ± 45 %) (Fig. 8 A-C, Table

2) compared to the saline control condition. However, the proportion of DOP positive neurons co-

expressing MOP receptors (90 ± 9 % vs 54 ± 12 %) and of MOP positive neurons co-expressing DOP

receptors (78 ± 13 % vs 28 ± 9 %) were significantly higher than in the saline control condition

(Fig.8 D-E, Table 2). Accordingly, a statistically significant increase in the proportion of

MOP/DOP positive neurons was detected compared to the saline condition (38 ± 6 % vs 17 ± 5

%) (Fig. 8F, Table 2).

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In abstinent animals, the density of DOP, MOP and DOP/MOP neurons were slightly

increased compared to the morphine-dependent or control animals (Fig. 8 A-C) and the

proportion of DOP positive neurons co-expressing MOP receptors (92 ± 4 %), MOP positive

neurons co-expressing DOP receptors (71 ± 8 %) and MOP/DOP positive neurons (40 ± 3 %) (Fig.

8 D-F, Table 2) remained similar to what we observed in morphine-dependent mice and

significantly higher than in the saline control condition.

Altogether, our results show different regulation of the MOP and DOP neuronal densities in the

LC/Bar nuclei or the RVM in morphine-dependent and abstinent mice. However, we observe in

both areas a strong increase in the percentage of MOP/DOP neurons after chronic morphine

administration that persisted after four weeks of abstinence.

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Figure 7. Impact of chronic morphine administration on DOP, MOP and MOP/DOP populations in the locus

coeruleus/Barrington nuclei. Density of (A) DOP, (B) MOP and (C) MOP/DOP neurons and relative proportion of (E) DOP,

(F) MOP, and (G) MOP/DOP neurons in saline, chronic morphine treated and abstinent mice. n=7 saline, n=4 chronic

morphine, n=6 abstinent mice. One-way ANOVA, Dunnett’s post-hoc analysis, *p<0.05, **p<0.01.

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Figure 8. Impact of chronic morphine administration on DOP, MOP and MOP/DOP populations in the rostral

ventromedial medulla. Density of (A) DOP, (B) MOP and (C) MOP/DOP neurons and relative proportion of (E) DOP,

(F) MOP, and (G) MOP/DOP neurons in saline, chronic morphine treated and abstinent mice. n=4 saline, n=4 chronic

morphine, n=5 abstinent mice. One-way ANOVA, Dunnett’s post-hoc analysis, *p<0.05.

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DISCUSSION Here, we examined the distribution of neurons co-expressing MOP-mCherry and DOP-eGFP

constructs in the brain of morphine-dependent and four week-abstinent mice to identify areas

where MOP/DOP heteromers can form. Our main finding indicates that chronic morphine

administration expands MOP/DOP neuronal co-expression throughout the brain with most

changes in MOP/DOP neuronal distribution still detectable after 4 weeks of abstinence.

Methodological considerations Detection of MOP/DOP receptor neuronal co-expression relies on the concomitant visualization of

the two fluorescent signals but also on our ability to fit their distribution with identifiable

neuronal structures. As previously published, DOP and MOP fluorescent constructs were detected

in all regions with previously identified wild type DOP or MOP receptor expression (4). However,

DOP and MOP receptor expression levels range from 10 to 60 fmol/mg in most brain regions (21-

24), which raises the possibility of overlooking areas with the lowest expression levels. Visualization

of MOP-mCherry is easy owing to the intracellular accumulation of the red fluorescence even in

low expressing neurons and does not require amplification. On the contrary, the green

fluorescence associated with the DOP receptor is often weak and needs therefore to be amplified

with eGFP-specific antibodies. In addition, identification of neuronal cell bodies has been improved

by treating animals with the DOP agonist SNC 80 that concentrates the green fluorescence in the

soma in a manner similar to MOP-mCherry as previously described (4). Combining

fluorescence amplification with agonist treatment significantly enhanced the sensitivity of our

approach and drastically improved identification of DOP-eGFP neurons and, hence, MOP/DOP

positive neurons. Nevertheless, the proportion of detected MOP/DOP positive neurons likely

represents a low estimate of the population co-expressing MOP and DOP receptors. In addition,

whether identification of novel brain areas in morphine-treated animals is linked to

transcriptional changes or rather reflects increased receptor expression, thereby enabling to

overcome the limit of detection, remains to be determined.

Chronic morphine administration expands MOP/DOP neuronal co-expression MOP/DOP neuronal co-expression was maintained in the 44 brain areas previously detected in

the basal state (4). These regions are part of neuronal networks essential for survival including

feeding and sexual behaviours but also those relevant to motor activity, perception and

processing of aversive stimuli such as pain (4). Our study also shows that the percentage of

MOP/DOP neurons among MOP and DOP expressing populations slightly varies according to the

brain area considered. They represent about 20 % of the neurons expressing MOP or DOP receptors

in the RVM, the CA1 and CA3 areas where functional MOP/DOP heteromers have been

identified (4, 19, 25). However, the proportion of MOP/DOP neurons is only about 10 % in

the DG and the LC/Bar nuclei.

In morphine-dependent animals, MOP/DOP neuronal co-expression significantly expands. One

important increase can be observed in the motor circuit which gets along with morphine-

increased locomotion (26). Similarly, more MOP/DOP neurons were identified in the reward.

pathway including the VTA, where ligand occupancy of the two receptors synergistically increased

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hyperpolarization in a subset of neurons (9). Therefore, decreased inhibitory tone on

dopaminergic cells is expected to facilitate dopaminergic release and to stimulate the

mesolimbic pathway underlying drug anticipation/reward (27). This clearly suggests that

MOP/DOP neurons, and very likely heteromers, contribute to the neurobiological changes

underlying opiate addiction. As expected, enhanced MOP/DOP neuronal co-expression is

detected in brain areas associated with somatic and vegetative symptoms of withdrawal such as the

parabrachial nucleus, medullary reticular areas, salivatory nucleus and solitary nucleus (28). Our

data thus provide a neuroanatomical support to explain why selective targeting of MOP/DOP

heteromers with the bivalent ligand MDAN-21 (5) or the biased agonist CYM51010 (14) led

to reduced withdrawal symptoms compared to morphine therefore pointing to MOP/DOP

heteromers as a valuable target for opiate maintenance. Another remarkable feature is the

enhanced detection of MOP/DOP neurons in the forebrain where they are scarce under

basal conditions. The neuronal circuits to which these new areas belong, such as the extended

amygdala, the dorsal/ventral basal ganglia or the septal circuits, are associated with emotional

processing (29). Interestingly, enhanced MOP/DOP co-localization in the forebrain correlates with

a similar MOP/DOP increase in brainstem structures with which they are connected, further

delineating neuronal circuits that tightly connect autonomous/visceral functions with

emotional/aversive processing (30, 31). For example, the insular cortex, central amygdala, ventral

pallidum, parabrachial nucleus and nucleus of the solitary tract all contribute to the expression

of conditioned taste aversion (32). Moreover, MOP/DOP forebrain structures are involved in

affective and motivational aspects of withdrawal (33-35) as well as in withdrawal memories (36).

Altogether, MOP/DOP neuroanatomical distribution confirms their therapeutic potential in the

context of opiate addiction.

Changes in MOP/DOP neuronal co-expression persist in the abstinent state A major finding of our study is the high extend by which MOP/DOP neuronal co-expression elicited

by chronic morphine persists in the abstinent state, which designates MOP/DOP neurons in

these pathways as biomarkers of previous repeated exposure to opiates. Indeed, close to 70% of

the novel areas identified in morphine-dependent mice are still detected four weeks after the last

drug injection. The highest prevalence of persisting changes was observed in circuits associated

with somatic (e.g LC and its noradrenergic input, the paragigantocellular nucleus) and visceral

(e.g.solitary complex, inferior salivatory nucleus) symptoms of opiate withdrawal described in

morphine-dependent animals above (30-32) as well as its emotional processing. For example,

MOP/DOP co-expression persisted in the central amygdala, the main component of the

amygdaloid autonomic system that mediates many autonomic, somatic, endocrine and

behavioral responses (29) as well as the affective component of opiate withdrawal (33-35).

Our results clearly support a view according to which the abstinent state represents a

different homeostatic state from naïve animals with increased vulnerability to relapse (37).

Although physical dependence to morphine is no longer detectable after 4 weeks of protracted

abstinence in mice (38), this time-point has been characterized by a significant reduction in

social interaction and the appearance of depressive-like behaviors in mice following chronic

treatment with morphine (38) or heroin (39). This is in line with other previous data indicating

motivational consequences of withdrawal associated with increased emotional vulnerability (40,

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41) and higher sensitivity to sensory input (33). It also reinforces our conclusions drawn in

morphine-dependent animals pointing to MOP/DOP as a crucial actor in opiate withdrawal, as

their selective pharmacological targeting led to reduced withdrawal symptoms (5, 14).

Increased MOP/DOP co-expression is also to be considered in the context of opiate induced

hyperalgesia often observed in patients on opiate maintenance treatment (42, 43).

Electrophysiological data support an antinociceptive role for MOP/DOP heteromers in the RVM.

Indeed, co-injection of subthreshold doses of the MOP agonist DAMGO and the DOP agonist

deltorphin II in the RVM of rats chronically treated with morphine decreased GABAergic

neurotransmission through synergistic activation (10), thereby reducing the GABAergic inhibitory

tonus exerted on the descending antinociceptive pathways.

Conclusion Altogether, our study establishes broader MOP/DOP neuronal co-expression following

chronic morphine that largely remains in the abstinent state. Persistence of MOP/DOP neuronal co-

expression in neuronal circuits mainly related to somatic symptoms of drug withdrawal but also

involved in its emotional processing or visceral sensory perception identifies MOP/DOP

distribution as a biomarker of opiate addiction. It also points to a role for MOP/DOP heteromers in

the modulation of relapse and hyperalgesia that designate them as an innovative therapeutic

target.

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