Opioid receptor gene expression in the rat brain during ontogeny, with special reference to the...

Ž .Developmental Brain Research 109 1998 187–199

Research report

Opioid receptor gene expression in the rat brain during ontogeny, withspecial reference to the mesostriatal system: an in situ hybridization

study

Francois Georges ), Elisabeth Normand, Bertrand Bloch, Catherine Le MoineCentre National de la Recherche Scientifique, Unite Mixte de Recherche 5541, Laboratoire d’Histologie Embryologie, Institut Federatif de Recherche en´ ´ ´

Neurosciences Cliniques et Experimentales, UniÕersite Victor Segalen Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France´ ´ ´ ´

Accepted 12 May 1998

Abstract

The three main types of opioid receptors m, d and k are found in the central nervous system and periphery. In situ hybridization studywas undertaken to determine the expression of m, d, k-opioid receptors mRNAs in the brain during pre- and postnatal development,especially in the mesostriatal system. By G13, m and k-opioid receptor mRNA were detectable in the telencephalon; m-opioid receptormRNA was found in the striatal neuroepithelium and cortical plate and k-opioid receptor mRNA in the corroidal fissure. By G15,k-opioid receptor mRNA was detectable in the nucleus accumbens and dorsal striatum, and in the substantia nigra and ventral tegmentalarea, suggesting an early expression of the corresponding receptor on dopaminergic terminal fibers. For the m-opioid receptor mRNA inthe striatum, patches appeared at G20. d-Opioid receptor mRNA was first detected at G21, in many areas including the accumbensnucleus and the dorsal striatum. At P8, d-opioid receptor mRNA was detected in large-sized cells of the striatum, possibly cholinergic,suggesting a possible modulation by opioids of the striatal cholinergic neurons. Our results demonstrate the early appearance of m and

Ž . Ž .k-opioid receptor mRNA G13 and the relatively late development of d-opioid receptor mRNA G21 in the brain. We also show adistinct pattern of expression for m, d and k-opioid receptor mRNAs in the mesostriatal system during the development. q 1998 ElsevierScience B.V. All rights reserved.

Keywords: Development; Mu; Delta; Kappa; Opioid receptor; Striatal compartment

1. Introduction

The classification of opioid receptors in at least threeŽ . Ž .pharmacologically distinct types, mu m , delta d and

Ž . w xkappa k , is now well established 4,25,28 . With thedevelopment of selective agonists and antagonists, it hasbecome clear that each type of opioid receptor has specific

w xpharmacological properties 12 , is differentially dis-w xtributed in the central nervous system 27,30 , and is

involved in a broad range of behaviors and functions,including regulation of pain, reinforcement and reward,release of neurotransmitters, as well as neuroendocrine

w xmodulation 2,38,42 . Endogenous opioids, and opioid re-ceptors, known as the endogenous opioid systems, havebeen essentially described during postnatal ontogeny

) Corresponding author. Fax: q33-5-56-98-61-82; E-mail:[email protected]

w x6,18,19,29,37,41,53 . In the adult, pharmacological stud-ies suggest that opioid receptors are involved in reward,motivational and tolerance processes, especially in relation

w xto the mesostriatal dopaminergic system 47 . Early on,several markers including dopamine innervation, opioidreceptors, acetylcholinesterase, D1 dopamine receptors andthe phosphoprotein DARPP-32, form patches in the cau-

w xdate–putamen and the nucleus accumbens 3,9,15,34,35 .The progression of events leading to mature striatalchemoarchitecture is not over by birth. It has been clearlyshown that the adult striatum is an heterogeneous structurecomposed of two main compartments, the patches and the

w xmatrix 11,14 . The development of the patches in thecaudate–putamen is a particularly intriguing process withthe m-opioid receptor distribution changing from a diffuseto a patchy pattern in the perinatal rat caudate–putamenw x49 . The cloning of the different opiate receptor genes hasprovided a sensitive tool to identify the distribution and the

0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0165-3806 98 00082-0

( )F. Georges et al.rDeÕelopmental Brain Research 109 1998 187–199188

cellular localization of the corresponding mRNAs in thew xadult brain by in situ hybridization 26 . Until now,

anatomical information on opiate receptors during develop-

ment, has been restricted to binding studies mainly duringw xpostnatal development 6,17,19,41,44,52 . Indeed the dif-

ferent opioid receptor binding sites showed distinct onto-

Fig. 1. Detection of the m-opioid receptor mRNA in the forebrain during pre- and postnatal ontogeny and in the adult. Images of X-ray films after in situ35 Ž .hybridization with S labeled cRNA probe. m-Opioid receptor mRNA was detected in the striatal anlage St from G13 onwards. G14–G16: m-opioid

Ž . Ž .receptor mRNA appeared all along the basal telencephalon Btel and in the striatal anlage St . G17–G19: m-opioid receptor mRNA shows aŽ . Ž .homogeneous distribution within the caudate–putamen CP and nucleus accumbens NAc . G20–G21: the labeling became heterogeneous and was patchy

Ž . Ž .at birth and at all postnatal stages studied. On and after G16, m-opioid receptor mRNA was detected in the cortex Cx , layer VIb and in the septum SP .Ž . 35LVs lateral ventricule . Control shows the nonspecific labeling after in situ hybridization with S labeled sense cRNA probe at P9. For pre- and postnatalstages, scale bars2.4 mm. In the adult, m-opioid receptor mRNA shows a patchy distribution within the caudate–putamen and nucleus accumbens andm-opioid receptor mRNA was also detected in the cortex, and in the septum. Scale bars4.5 mm.

( )F. Georges et al.rDeÕelopmental Brain Research 109 1998 187–199 189

genic patterns, with m and k binding sites being presentbefore birth, but d binding sites emerging postnatally. Insitu hybridization and immunohistological studies havereported the presence of proenkephalin gene and peptide at

w xembryonic day 16 in the striatal anlage 43 and dynorphinw xpeptide in both pre- and postnatal period 41 , suggesting a

possible action on opiate receptors. However, binding stud-ies detected both presynaptic and postsynaptic receptors,with no information about their sites of synthesis. Onequestion is whether these binding sites can be closelyrelated to the distribution of m, d and k-opioid receptormRNA in the pre- and postnatal periods of development.Our study was undertaken to describe the expression of m,d, k-opioid receptor mRNAs in the rat brain, and morespecifically in the mesostriatal system, during pre- andpostnatal development by using in situ hybridization.

2. Material and methods

2.1. Animals and tissue preparation

Ž .Timed pregnant rats, IOPS OFA Iffa-Credo France ,sperm positive on a specific day, were used. Gestationalday G1 designated as the day following a successfulinsemination. P0 was the day of birth after a gestation of21 days. Prenatal series were obtained from embryos andfetuses at ages G13 to G21 and postnatal series from

Ž .animals at ages P1, P2, P4, P6, P8, P9 ns2 per stage .Ž .Brains of adult male rats ns5 were used as controls.

The pregnant rats and the postnatal pups were anesthetizedwith chloral hydrate. Subsequently, embryos and fetuseswere removed and dissected out free from the amnioticmembranes. For the stages G17 to P9, the brains were

Fig. 2. Detection of the m-opioid receptor mRNA in the diencephalon during pre- and postnatal ontogeny. Images of X-ray films after in situ hybridization35 Ž .with S labeled cRNA probe. On and after G19, high levels of m-receptor mRNA expression were observed in the medial habenula Mhb and in several

Ž . Ž . Ž . Ž .thalamic nuclei, including paraventricular PV , centromedial CM , rhomboid RH , ventral posterolateral and ventral posteromedial VPLr VPMŽ . Ž .paracentral PC nuclei. In the hypothalamus, cells expressing m-receptor mRNA were prominent in several nuclei including ventromedial VMH and

Ž . Ž .arcuate Arc nuclei. m-Opioid receptor mRNA was also found in amygdala Amy . Scale bars2.4 mm.


dissected out, fixed 24 h by immersion in 1 or 4%Ž .paraformaldehyde PFA then cryoprotected by immersion

in 15% sucrose phosphate buffer for 24 h. The wholeŽ .heads of the embryos G13–G16 were fixed as above.

Tissues were cut in 10 mm cryostat serial coronal sectionsthat were collected on gelatin-coated slides and stored aty808C until use.

2.2. In situ hybridization

Radiolabeled antisense cRNA probes were synthesizedby in vitro transcription from plasmid constructsŽ w .pBluescript, Stratagene containing cDNAs encoding rat

Ž w x. Žm fragment 744–1064 bp 48 , d fragment 304–1287 bpw x. Ž w x.26 and k fragment 1351–2124 bp 31 . The transcrip-

Fig. 3. Detection of the k-opioid receptor mRNA in the forebrain during pre- and postnatal ontogeny and in the adult. Images of X-ray films after in situ35 Ž .hybridization with S labeled cRNA probe. k-Opioid receptor mRNA were first detectable at G13 in the corroidal fissure CF . At G15, k-opioid receptor

Ž .mRNA was detected in the striatal anlage St . On and after G17, cells expressing k-opioid receptor mRNA were localized in the lateral area of theŽ . Ž . Ž . Ž .caudate–putamen CP , and in the nucleus accumbens NAc . k-Opioid receptor mRNA was also found in endoperiform nucleus En , claustrum Cl andŽ . 35olfactory tubercle TO . Control shows the nonspecific labeling after in situ hybridization with S labeled sense cRNA probe at P8. For pre- and postnatal

stages, scale bars2.4 mm. In the adult, k-opioid receptor mRNA was localized in dorsal and ventral parts of the striatum. k-Opioid receptor mRNA wasalso found in endoperiform nucleus, claustrum, and olfactory tubercle. Scale bars4.5 mm.


tion was performed from 50–100 ng of linearized plasmidw35 x Ž .using S UTP 1300 Cirmmol; NEN and T3 or T7 RNA

Ž .polymerase GibcoBRL . After alkaline hydrolysis to ob-tain 250 base pair-cRNA fragments, the probes were puri-fied on Sephadex G50 column and precipitated in sodium

Ž . Ž .acetate 0.1 vol. rabsolute ethanol 2.5 vol. . The proce-dure of in situ hybridization employed here has been

w xoriginally described by Le Moine and Young 24 and wasused here with minor modifications. Briefly, the sectionswere fixed with 4% PFA for 5 min at room temperature,rinsed twice in 4= sodium chloride–sodium citrate bufferŽ .SSC , acetylated into 0.25% acetic anhydride in 0.1 M

Ž .triethanolaminer4= SSC pH 8 for 10 min at roomtemperature and then dehydrated in graded alcohol. The

ŽcRNA probes were diluted in hybridization buffer 20 mMTris–HClr1 mM EDTAr300 mM NaClr50% for-mamider10% dextran sulfater1 = Denhardt’sr250mgrml yeast tRNAr100 mgrml salmon spermDNAr0.1% sodium dodecyl sulfater0.1% sodium thiosul-

.fate to a final concentration of 0.7 mCir50 ml correspond-ing to 1–2 ng of 35S-labeled cRNA probe. Fifty microlitersof probe were applied to the brain sections and cover-

slipped. After an overnight hybridization at 558C, theŽ .slides were rinsed in 4= SSC 4=5 min and treated with

Ž .RNAse A 20 mgrml for 20 min at 378C. The slides wereŽ . Žthen washed with 2= SSC 5 min, twice , 1= SSC 5

. Ž .min , 0.5= SSC 5 min at room temperature, and rinsedŽ .in 0.1= SSC at 658C 30 min, twice before dehydration

in graded alcohols, and air dried. In parallel, controls ofspecificity were performed in the same conditions withsense cRNA probes for each receptor. The slides were then

Ž .exposed to Biomax film Kodak for 20 days and dipped inIlford K5 emulsion, exposed for 10–12 weeks at 48C,developed, and counterstained with Mayer’s hemalun. Allpre- and postnatal stages sections were performed in thesame experiment.

2.3. mRNA quantitation

Quantitative measurements of the mRNA levels wereperformed by autoradiographic densitometry with a Bio-

Ž .com 200 image analyser Les Ulis, France . Radioactivestandards were generated using brain paste. A calibration

Ž .curve representing optical densities OD as a function of

Fig. 4. Detection of the k-opioid receptor mRNA in the diencephalon during pre- and postnatal ontogeny. Images of X-ray films after in situ hybridization35 Ž .with S labeled cRNA probe. On and after G19, k-opioid receptor mRNA expression was limited to the lateral and medial habenula LHb and MHb , and

Ž . Ž . Ž .to some nuclei of the thalamus such as the paraventricular PV , dorsal lateral geniculate DLG and posterior Po nuclei. In the hypothalamus, cellsŽ . Ž . Ž . Ž .expressing k-opioid receptor mRNA were prominent in several nuclei including the dorsomedial DM , posterior PH , arcuate Arc , ventromedial VMH

Ž . Ž . Ž .nuclei and mammillothalamic tract mt . Cells expressing k-receptor were also observed in the amygdaloid nuclei Amy and piriform cortex PIR . Scalebars2.4 mm.


Fig. 5. Detection of the k-opioid receptor mRNA in the mesencephalon during pre- and postnatal ontogeny and in the adult. Images of X-ray films after in35 w xsitu hybridization with S labeled cRNA probe. At G15, k-opioid receptor mRNA were observed in the dopaminergic nuclear complex A9–A10 . From

Ž . Ž . Ž .G19 on, cells expressing k-receptor mRNA were localized in the substantia nigra SN , ventral tegmental area VTA , central gray CG , superiorŽ . Ž . Ž . Ž . Ž . Ž .colliculus SC red nucleus R , pineal gland Pi , posterior commissure pc , nuclei optic tract OT , amygdalo hippocampal area posteromedial AHPM

Ž .and medial geniculate nuclei MG . For pre- and postnatal stages, scale bars2.4 mm. In the adult, k-opioid receptor mRNA was localized in theŽ .substantia nigra, ventral tegmental area, central gray, superior colliculus, amygdalo hippocampal area posteromedial and occipital cortex Oc . Scale

bars4.5 mm.

Fig. 6. Detection of the d-opioid receptor mRNA in the forebrain during peri- and postnatal ontogeny and in the adult. Images of X-ray films after in situhybridization with 35S labeled cRNA probe. d-Opioid receptor mRNA was first detected at G21, and from G21 to P9 showed a homogeneous distribution

Ž . Ž . Ž .within the caudate–putamen CP and nucleus accumbens NAc . At all stages studied, d-opioid receptor mRNA was detected in the cortex Cx . Controlshows the nonspecific labeling after in situ hybridization with 35S labeled sense cRNA probe at P9. For pre- and postnatal stages, scale bars2.4 mm. Inthe adult, d-opioid receptor mRNA showed a homogeneous distribution within the caudate–putamen and nucleus accumbens. In the cortex, d-opioidreceptor mRNA expression was restricted to cortical layers II and V, and the superficial portion of layer VI. Scale bars4.5 mm.


the radioactivity concentration was calculated from thesestandards. The ODs were measured in the striatum and inthe corpus callosum, at all pre- and postnatal stages, and inthe adult. These values were representative of the quantityof probe hybridized, and therefore correspond to the rela-tive mRNA level. The results were expressed as ratio of

ŽOD OD in the striatumrOD in the corpus callosum,.OD rOD for each stage.Str cc

3. Results

Our data show that m, d and k-opioid receptor mRNAswere differentially distributed in the rat brain, especially inthe mesostriatal system during development. Each mRNA

has a specific time of appearance and a distinct anatomicaldistribution during pre- and postnatal development. Figs.1–7 show images of X-ray films for the distribution of m,d and k-opioid receptor mRNAs at different stages, in thestriatum and the mesencephalon, but also in others regionssuch as the thalamus, hypothalamus, amygdala, hippocam-pus and cortex. Images at the cellular level are shown inFig. 8. The reference for the anatomical nomenclature was

w xthe atlas of Paxinos et al. 36 .

3.1. Distribution of the m-opioid receptor mRNA

Fig. 1 shows the distribution of m-opioid receptormRNA in the forebrain at different stages of development.Specific hybridization signal for m-opioid receptor mRNA

Fig. 7. Detection of the d-opioid receptor mRNA in the diencephalon during peri- and postnatal ontogeny. Images of X-ray films after in situ hybridization35 Ž .with S labeled cRNA probe. On and after G21, d-opioid receptor mRNA expression was limited to some nuclei such as medial habenula Mhb and the

Ž .thalamic paraventricular nucleus PV . In the hypothalamus, cells expressing d-opioid receptor mRNA were observed essentially in the ventromedial nucleiŽ . Ž . Ž . Ž .VMH . Cells expressing d-receptor were prominent in the amygdaloid nuclei Amy , hippocampal formation Hi and the dentate gyrus DG . Controlshows the nonspecific labeling after in situ hybridization with 35S labeled sense cRNA probe at G21. Scale bars2.4 mm.


was detected on autoradiograms from G13 on. At thisstage, the striatal anlage exhibited distinct hybridizationsignal. At G14–G16, high levels of m-opioid receptormRNA appeared all along the basal telencephalon. At thisstage of development, the labeling in the striatal anlage

Ž .was intense and homogeneous. At later stages G17–G19 ,m-opioid receptor mRNA was observed throughout thecaudate–putamen and the nucleus accumbens, and theseareas showed a strong and homogeneous labeling. AtG20–G21, m-opioid receptor mRNA became distributed inpatches. At all postnatal stages studied, the general topog-

w xraphy and the patchy aspect of labeling Fig. 8A wasw xsimilar to that observed at the adult stage frame in Fig. 1 .

Within the patches, neurons are small, round and closetogether, which gives a dense and compact aspect of the

w xlabeling Fig. 8G . We observed that the overall intensityof labeling in the striatum decrease during development.Indeed, during prenatal ontogeny, the ratio OD rODStr cc

ranged from 16.1"0.64 at G14–G16 to 10.3"1.12 atG17–G19 and 8.65"0.17 at G20–G21. After the birth,the ratio ranged from 5.68"0.38 at postnatal stages anddecreased to 2.3 at adult stage. In the frontal, parietal andcingular cortex, cells expressing m-opioid receptor mRNAwere found from G17, in the deepest portion of layer VIŽ .VIb , and increase at G21 to P9. On and after G17,m-opioid receptor mRNA was detected in the septum. Nolabeling was observed on X-ray films in mesencephalicareas, but some neurons expressing m-opioid receptormRNA are seen in the ventral tegmental area and in thepars compacta of the substantia nigra at several postnatal

w xstages Fig. 8H .Specific hybridization signal for the m-opioid receptor

mRNA was also detected in the diencephalic area fromG13 on. At G15–G18, high levels of mRNA were ob-served in the thalamus, hypothalamus and amygdaloid

Ž .neuroepithelium data not shown . At G19–G21, highlevels of m-opioid receptor mRNA expression were de-tected in medial habenula and in most thalamic nucleiŽparaventricular, centromedial, rhomboid, and ventropos-

. w xterolateral thalamic nuclei Fig. 2 . In the hypothalamus,m-opioid receptor mRNA shows a widespread distributionbut high levels of expression were limited to a few nuclei

Ž . w xventromedial, arcuate Fig. 2 . At all postnatal stagesstudied, m-opioid receptor mRNA expression was observedin the same thalamic and hypothalamic nuclei than inadult, but the general intensity of hybridization signals waslower.

3.2. Distribution of the k-opioid receptor mRNA

Fig. 3 shows the expression of k-opioid receptor mRNAin the forebrain at different stages of development. k-Opioid receptor mRNA was detectable at G13 with anintense signal restricted to the corroidal fissure. At G15,k-opioid receptor mRNA was first detectable in the striatalanlage. At G17–G21, and all postnatal stages, the generaldistribution of k-opioid receptor mRNA in the striatumwas similar to the adult with an increasing gradient from

w xventromedial to dorsolateral areas frame in Fig. 3 . Highlevels of k-opioid receptor mRNA were observed in thenucleus accumbens, olfactory tubercle, claustrum and en-dopiriform nucleus. From G21, the k-opioid receptormRNA expression in the nucleus accumbens became het-erogeneous, with cells expressing k-opioid receptor mRNA

Žbeing organized into clusters double arrows at P2 and P8.in Figs. 3 and 8B and F . In the dorsal striatum k-opioid

receptor mRNA was principally localized in neurons of thedorsolateral portion of the caudate–putamen. k-Opioid re-ceptor mRNA was also detected in the claustrum, theendopiriform nucleus and the olfactory tubercle.

Specific hybridization signal for the k-opioid receptormRNA was detected in the diencephalic area from G13 onŽ .data not shown . Fig. 4 shows the expression of k-opioidreceptor mRNA in the diencephalon at different stages ofdevelopment from G19 where high levels of k-opioidreceptor mRNA were detected in most thalamic and hy-pothalamic nuclei. k-Opioid receptor mRNA was indeed

Ždetected in the thalamus in the areas of paraventricular,posterior, ventral postero lateral, ventral postero medial

.nuclei and in the medial habenula. In the hypothalamus,k-opioid receptor mRNA presented a strong labeling inventromedial, arcuate, lateral, posterior hypothalamic nu-clei. At postnatal stages, high levels of k-opioid receptormRNA were observed in the mammillary complex, amyg-

Ž . Ž .Fig. 8. Detection of m, d and k-opioid receptor mRNA at the cellular level in the caudate–putamen CP , nucleus accumbens Nac and substantia nigraŽ . 35 Ž . Ž .SN during pre- and postnatal ontogeny. In situ hybridization was performed with S labeled cRNA probe. A–D Dark-field photomicrographs. A atP4, m-opioid receptor mRNA showed a patchy distribution in the caudate–putamen and nucleus accumbens; note the labeling of the subcallosal streakŽ . Ž .arrows . B At P8, k-opioid receptor mRNA showed an intense and heterogeneous distribution in the nucleus accumbens, and also some labeling in the

Ž . Ž .caudate–putamen and in the claustrum Cl . C At P9, in contrast to the m and k-opioid receptor mRNA, cells expressing d-opioid receptor mRNA had amore homogeneous distribution in the caudate–putamen. The arrows indicated dispersed cells, probably cholinergic interneurons expressing d-opioid

Ž . Ž .receptor mRNA. D At G21, cells expressing k-opioid receptor mRNA showed an intense expression in the substantia nigra. E–I BrightfieldŽ .photomicrographs; at all postnatal stages studied, the neurons were small, round and close together, giving a dense and compact labeling. E At P4,

Ž .k-opioid receptor mRNA is expressed in neurons of the substantia nigra, pars compacta. F At P8, small neurons showed a k-opioid receptor mRNAŽ . Ž . Ž .labeling arrowheads in the caudate–putamen. Several unlabeled neurons were also present. G Detection of the m-opioid receptor mRNA in a patch P

Ž .of the caudate–putamen where the majority of the small neurons are labeled. H At P4, few cells expressing m-opioid receptor mRNA are seen in theŽ . Ž . Ž .substantia nigra, pars compacta arrowhead . I At P8, three large-sized neurons arrowheads expressing d-opioid receptor mRNA are visible in the

caudate–putamen. Scale barss0.5 mm in A and B, 0.1 mm in C, 0.2 mm in D and 10 mm in E to I.


dala nuclei and piriform cortex. During all ontogenic stagesstudied, the general topography of labeling in the dien-cephalon was equivalent to that observed at the adult stageŽ .data not shown .

Fig. 5 shows the expression of k-opioid receptor mRNAin the mesencephalon at different stages of development.From G15, k-opioid receptor mRNA was abundant in alarge group of cells near the pial surface at the mesen-cephalic flexure, corresponding to the developing

Ž .dopaminergic nuclear complex A9–A10 . At stages G19–G21, the A9–A10 complex separated into the substantia

Ž . Ž .nigra A9 and ventral tegmental nucleus A10 , bothwshowing a strong signal for k-opioid receptor mRNA Fig.

x8D . At all postnatal stages, the general topography oflabeling in the substantia nigra and the ventral tegmentalarea was similar to the adult: k-opioid receptor mRNA isexpressed in large cells with many characteristics of

Ždopaminergic neurons appearance, distribution ant fre-. w xquency Figs. 5 and 8E . Moreover, at all ontogenic

stages, k-opioid receptor mRNA were detected in thesuperior colliculus, central gray, medial geniculate nucleus,red nucleus, posterior commissure, amygdalo hippocampalarea postero medial and pineal gland.

3.3. Distribution of the d-opioid receptor mRNA

Fig. 6 shows the expression of d-opioid receptor mRNAin the forebrain at different stages of development. Spe-cific hybridization signal for d-opioid receptor mRNA wasfirst detected at G21. From G21 to P9, d-opioid receptormRNA expression in the nucleus accumbens andcaudate–putamen appeared homogeneous. This distribu-

wtion appears in a pattern similar in the adult frame in Figs.x6 and 8C . At P4–P9, a small proportion of large cells

localized in the dorsolateral part of caudate–putamen,possibly cholinergic, contained particularly high levels of

Ž .d-opioid receptor mRNA Fig. 8C,I . At all stages studied,d-opioid receptor mRNA expression in the neocortex wasrestricted to two broad bands, internal and external, sepa-rated by a thinner band of much lower d-opioid receptormRNA density most probably corresponding to corticallayers II and V, and the superficial portion of layer VI.

Fig. 7 shows the expression of d-opioid receptor mRNAin the diencephalon at different stages of development.d-Opioid receptor mRNA was detected throughout thethalamus and hypothalamus, for G21 to P9. d-Opioidreceptor mRNA expression was limited mainly to themedial habenula and paraventricular nucleus of the thala-mus. At peri- and postnatal stages, only low levels ofd-opioid receptor mRNA expression were observed in theventromedial hypothalamus, as well as the hippocampusand the dentate gyrus. At G21 and all postnatal stages, thegeneral distribution of d-opioid receptor mRNA in thala-mus and hypothalamus was similar to that of the adultŽ .data not shown .

4. Discussion

The present results describe the distribution of m, d,k-opioid receptor mRNA in the brain during pre- andpostnatal development. The aim of our study was to local-ize precisely the sites of synthesis for the different opioidreceptors at early stages of development. Because of thefunctional importance of dopamineropioids interactions inthe striatonigral pathway, we will focus the discussion onthe expression of m, d, k-opioid receptor mRNA in thestriatal anlage and in the dopaminergic nuclear complexw xA9–A10 , representing the developing substantia nigraand ventral tegmental area. The present data bring evi-dence for opioid receptor gene expression in the brain,especially in the mesostriatal system, during pre- andpostnatal development. In accordance with binding studiesw x6 , we show here that m and k-opioid receptor mRNAwere detectable in the brain from G13, suggesting that m

and k are expressed and functional at this stage. Bycontrast, d-opioid receptor mRNA was detectable first atG21. These results show that the opioid receptors areexpressed early during ontogeny, and are likely to partici-pate to the development of the nervous system, particularlythe mesostriatal system.

4.1. m , d , and k-opioid receptor gene expression in thestriatum during pre- and postnatal ontogeny

Our results demonstrate the expression of m and k-opioid receptor mRNA in the striatal anlage as early asG13, and the first appearance of d-opioid receptor mRNAat G21. From G20 to P9, the m and k-opioid receptormRNA distribution in the striatum is similar to what isobserved in adults. For the m-opioid receptor mRNA,patches were detected in the caudate–putamen and nucleusaccumbens, whereas the k-opioid receptor mRNA labelingis mostly observed in the nucleus accumbens.

The distribution of d-opioid receptor mRNA at allpostnatal stages studied is similar to that of the adult.During the developmental stages studied, a moderate levelof d-opioid receptor mRNA expression is observed in mostneurons of the caudate–putamen and the nucleus accum-bens. At P4–P9 our results confirm the expression ofd-opioid receptor mRNA in cholinergic neurons found in

w xthe striatum of adult mice 23 . Indeed, d-opioid receptormRNA is detected in large-sized cells with characteristicoval or polygonal shaped soma which, considering theirdistribution and their morphological characteristics, arelikely to be cholinergic interneurons. Striatal choline acetyltransferase immunoreactive cells are born between G12and G17 and are among the earliest neurons to be gener-

w xated in the striatum 39 . The depolarization-induced re-lease of acetylcholine is inhibited by activation of d recep-

w xtor 33,44 . Our present results demonstrate the presence ofd-opioid receptor in cholinergic neurons mRNA as early as


P4, in agreement with results obtained by De Vries et al.w x7 . Indeed, these authors have demonstrated that a d-opioidreceptor agonist was already able to inhibit the release ofacetylcholine by about 60% at P7.

We show here that m-opioid receptor mRNA is moreabundant during prenatal ontogeny than in adult. Thestriatal neuroepithelium presents a strong intensity of sig-nal from G13, with a peak at G18–G19, which progres-sively declines after birth. It is known that the develop-ment of the striatum involves multiple cellular processes toachieve its mature cytoarchitecture. The distribution ofm-opioid receptor mRNA appears diffusely in the striatum

Ž .during the mid gestation G13–G19 , and becomes dis-tributed in patches at peri- and postnatal stages. These datamatch with the development of striatal compartmentationwhich follows these two successive patterns from homoge-neous to patchy. Indeed the pattern of m-opioid receptormRNA labeling is consistent with binding studies: patches,

w xmarked by high densities of opiate receptors 16,22 , firstemerge perinatally from a dense and diffuse field of

w xreceptor binding 49,50 . It seems that the high density ofm-opioid receptors detected in fetal rodent brain decreases

w xduring the postnatal period 1,6,43 . Developmental studieshave shown that the neurons of the striatal patch compart-ment become post mitotic primarily between G12 and G15w x43 and form striatonigral contact by G17. Striatal matrixneurons become post mitotic primarily between G17 and

w xG20 50 and do not form striatonigral connections untilthe first postnatal week. One of the cellular mechanismsunderlying striatal compartmentation appears to be the

w xselective self-adhesion of the patch neurons 20 . Thedevelopmental stage where patch neurons become selec-tively adhesive to each other is unknown, but this adhesivephenotype may be expressed in vivo soon after patchneurons were postmitotic. In agreement with this, it islikely that the m-opioid receptor mRNA expressing neu-rons reported here as early as G13 and during all ontogenicstages, correspond to the patch neurons, as described after

w xinjection of true blue into the substantia nigra 10,21,50 .This prenatal injection produced selective retrograde label-ing of early projecting patch neurons that clearly delin-eated the patches vs. the matrix compartment in the adultstriatum. Furthermore, during development, dopamine flu-orescence shows a patch-like distribution overlapping opi-

w xoid receptor binding sites in the rat striatum 34 .Our results also demonstrate that k-opioid receptor

mRNA was detectable at G15 in the striatal anlage andfrom G17, were localized in both dorsal and ventral partsof the striatum. In agreement, results obtained by radio-im-

w x w xmuno assay 41 and binding studies 1,44,52 report that m

and k-receptors as well as the dynorphin peptide werefound in both pre- and postnatal period. In situ hybridiza-tion and immunohistological studies have reported thepresence of proenkephalin gene and peptide on embryonic

w xday 16 in the striatal anlage 43 , suggesting a possibleaction on opiate receptors.

4.2. m and k opioid gene expression in the substantianigra and Õentral tegmental area

In the present study, we show the prominent presenceof k-opioid receptor mRNA in the rat embryonic dopamin-

Ž .ergic complex A9–A10 at G15. From G19, the presenceof k-opioid receptor mRNA in substantia nigra and ventraltegmental area suggests a localization in dopaminergicneurons. In contrast during postnatal development, rela-tively few cells expressing m-opioid receptor mRNA areseen in the ventral tegmental area and in the substantianigra pars reticulata. In agreement, adult lesion studiesdemonstrate a transport of opiate and dopaminergic recep-

w xtors into the dopaminergic fibres 40,51 , and pharmaco-logical studies show that k agonists selectively decreasepresynaptic dopamine release in the rat striatumw x8,33,45,46 . Our results suggest that the dopaminergicsystem may be modulated by the opiate system in themesostriatal system from G15. Indeed, previous studieshave reported that dynorphin, the k endogenous ligandw x5,13 as well as the k-receptor were also found in both the

w xpre- and postnatal period 19,41,44,52 . Electrophysiologi-cal data also demonstrate that k-opioid receptor are func-tional at G17, and are already able to strongly inhibit theelectrically evoked release of dopamine from striatal slicesw x7 . Therefore, our results extend previous studies andsuggest that the k-opioid receptor may play a role in braindevelopment in interaction with dopaminergic system.

5. Conclusion

Ž .In conclusion, our study demonstrates: 1 the earlyappearance of m and k-opioid receptors mRNA and therelatively late development of d-opioid receptor mRNA in

Ž .the brain; 2 that the two successive pattern of expressionŽof the m-opioid receptor gene from homogeneous to

.patchy are concomitant with the development of striatalŽ .compartmentation; 3 the prenatal expression of k-opioid

receptor mRNA in the A9–A10 complex, suggesting anearly expression on the dopaminergic terminals fibres.

Many data have provided evidence that opioid receptorsare functional during the perinatal period, and have thecapacity to modulate developmental processes such as

w xcellular proliferation and neuronal death 19,32,53 . Inaddition, these receptors may mediate possible develop-mental disturbances in these neurotransmission processesdue to prenatal exposure to opiates. However, the functionof opiate receptors during ontogeny is still relatively un-known and will require more detailed analysis. Furtherstudy of opioid receptor-deficient mice during the develop-ment will open new perspectives for understanding thefunctional roles of these receptors.


Acknowledgements

The authors thank Dr S.J. Watson and Dr A. Mansourfor the gift of the opioid gene receptor cDNA clones. Wealso thank C. Vidauporte for expert photographic artwork,M. Manse for excellent technical assistance and M. Jaberfor helpful comments on the manuscript.

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