Brain Research 943 (2002) 68–79www.elsevier.com/ locate /bres

Research report

Q uantitative autoradiography of adenosine receptors andNBTI-sensitive adenosine transporters in the brains and spinal cords of

mice deficient in the m-opioid receptor genea b b a aAlexis Bailey , Hans Matthes , Brigitte Kieffer , Susan Slowe , Susanna M.O. Hourani ,

a ,*Ian KitchenaPharmacology Group, School of Biomedical and Life Sciences,University of Surrey, Guildford, Surrey GU2 7XH, UK

b ´UPR 9050 CNRS, ESBS Universite Louis Pasteur, 67400 Illkirch, Strasbourg, France

Accepted 5 March 2002

Abstract

There is a large body of evidence indicating important interactions between the adenosine and opioid systems in regulating pain at boththe spinal and supraspinal level. Mice lacking the m-opioid receptor (MOR) gene have been successfully developed and the animals showcomplete loss of analgesic responses to morphine as well as differences in pain sensitivity. To investigate if there are any compensatoryalterations in adenosine systems in mutant animals, we have carried out quantitative autoradiographic mapping of A and A adenosine1 2A

receptors and nitrobenzylthioinosine (NBTI) sensitive adenosine transporters in the brains and spinal cords of wild type, heterozygous andhomozygous m-opioid receptor knockout mice. Adjacent coronal sections were cut from the brains and spinal cords of 1 /1, 1 /2 and

3 3 32 /2 mice for the determination of binding of [ H]DPCPX, [ H]CGS21680 or [ H]NBTI to A and A adenosine receptors and1 2A

3 3NBTI-sensitive adenosine transporters, respectively. A small but significant reduction in [ H]DPCPX and [ H]NBTI binding was detectedin mutant mice brains but not in spinal cords. No significant change in A binding was detected in m-opioid receptor knockout brains.2A

The results suggest there may be functional interactions between m-receptors and A adenosine receptors as well as NBTI-sensitive1

adenosine transporters in the brain but not in the spinal cord. 2002 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Opioid receptors

Keywords: m-Knockout; A receptor; A receptor; NBTI-sensitive adenosine transporter; m-Opioid receptor; Autoradiography; Knockout mice1 2A

1 . Introduction adenosine metabolism, greatly enhanced the spinal antial-lodynic effects of i.t. administered morphine in a rat model

There is a large body of evidence indicating that both of neuropathic pain in an additive manner [32]. Morphineacute and chronic effects of opioids are partly mediated by was shown to release adenosine from in vitro and in vivoadenosine [43]. Antinociception induced by intrathecal spinal cord preparations [55,56]. This release occurs from(i.t.) or intracerebroventricular (i.c.v.) administration of primary afferent nerve terminals in the dorsal horn of themorphine was shown to be inhibited by i.t. administration spinal cord [56] via nitrobenzylthioinosine (NBTI)-sensi-of the non-selective adenosine receptor antagonist theo- tive adenosine transporters [58]. Further, supraspinallyphylline in the tail flick assay in mice [9,10]. In addition, a administered morphine enhances purine release in therecent study demonstrated that i.t. administered adenosine cortex in vitro and in vivo [16,40,54] and intracerebroven-or i.t. administered adenosine kinase, which would block tricular morphine has also been shown to release adenosine

and adenosine 39–59-cyclic monophosphate from the spinalcord via a serotonergic mechanism [57]. The above studies*Corresponding author. Tel.: 144-1483-689-734; fax: 144-1483-576-not only suggest a local opioid–adenosine interaction at978.

E-mail address: i.kitchen@surrey.ac.uk (I. Kitchen). the level of the spinal cord, but also give evidence for

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )02536-2

A. Bailey et al. / Brain Research 943 (2002) 68 –79 69

spinal interactions with supraspinal opioid stimulated described in detail elsewhere [37]. All mice studied weredescending antinociceptive pathways. between 26 and 27 weeks of age and of mixed sexes.

In addition to its involvement in the expression of opioid There were no obvious differences between males andmediated analgesia [29,30,32,43,44], a role for adenosine females and data from both sexes were pooled.in the development of opioid tolerance, dependence andwithdrawal has also been suggested. While recent studies

2 .2. Autoradiographic procedurestend to agree that adenosine receptor agonists attenuate andantagonists exacerbate opioid withdrawal signs in the

General procedures for quantitative autoradiographycentral nervous system (CNS) of mice and rats [28,42,67],were performed as detailed previously [31]. Adjacentstudies suggesting an adenosine involvement in opioidfrozen coronal sections (20 mm thick) were cut at 300 mmtolerance have yielded contradictory findings [1,7,21].intervals throughout the brains and the spinal cords of wildHowever, cross-tolerance and cross-dependence betweentype (1 /1), heterozygous (1 /2) and homozygous (2 /2)m-opioid, A adenosine and a adrenergic antinociception1 2

m-knockout mice for the determination of total andin the periphery has been shown, suggesting a physical3non-specific binding of [ H]1,3-dipropyl-8-cyclopentyl-receptor interaction in the membrane or an interaction at

3xanthine (DPCPX), [ H]2-[ p-(2-carbonylethyl)phenyleth-the level of second messengers [3].ylamino]-59-N-ethylcarboxamidoadenosine (CGS21680)Some studies have demonstrated that interactions be-

3and [ H]nitrobenzylthioinosine (NBTI) to A adenosinetween chronically administered opioids and adenosine 1

receptors, A adenosine receptors and NBTI-sensitiveanalogues reflect changes in central adenosine receptor and 2A

adenosine transporters, respectively. Sections from 1 /1,transporter expression. Chronic exposure to morphine has1 /2 and 2 /2 animals were processed together. Ligandbeen demonstrated to upregulate A receptors in cortex1

concentrations were approximately 3–4 times K with[27] and brain homogenates [2] and NBTI-sensitive adeno- d3[ H]DPCPX used at a concentration of 3 nM,sine transporter binding sites in the striatum and hypo-3 3[ H]CGS21680 at 10 nM and [ H]NBTI at 4.5 nM. Non-thalamus of mice [26] and to downregulate adenosine A2A

specific binding was determined in the presence of 1 mMreceptors in striatum [8] and A receptors in the spinal16 3N -cyclopentyladenosine (CPA) for [ H]DPCPX, 20 mMcord of rats [59,60]. In contrast, other groups have shown

59-N-ethylcarboxamidoadenosine (NECA) forno change in A and A receptor numbers in cortex and1 2A3 3[ H]CGS21680 and 10 mM unlabelled NBTI for [ H]NBTIstriatum of chronic morphine-treated rats and mice, respec-

3 3binding. Slides used for [ H]CGS21680 and [ H]NBTItively [27,59].binding were pre-incubated for 30 min in 170 mM Tris–To ascertain further the involvement of the adenosineHCl plus 1 mM ethylenediaminetetraacetic acid (EDTA)receptors and transporters in mediating opioid effects, weand 50 mM Tris–HCl (pH 7.4 at room temperature),examined by quantitative autoradiography if there are anyrespectively. Then, incubation of slides withchanges in the binding and/or distribution of A or A1 2A

3 3[ H]CGS21680 and [ H]NBTI was carried out in eitherreceptors as well as NBTI-sensitive adenosine transportersTris–HCl (170 mM, pH 7.4) plus MgCl (10 nM, pH 7.4)in the brains and spinal cords of heterozygous (1 /2) and 2

or Tris–HCl (50 mM, pH 7.4) for 120 and 15 min,homozygous (2 /2) m-opioid receptor knockout mice3compared to wild type (1 /1). The generation of mice respectively. [ H]DPCPX binding was carried out in Tris–

deficient in the m-opioid receptor (MOR) gene has recently HCl (170 mM, pH 7.4) for 120 min. 2 U/ml of adenosinebeen reported by us and several other groups [37,53,62]. deaminase type VIII was added to all incubation buffers inThe knockout mice exhibited complete loss of analgesic order to remove endogenous adenosine. Following the

3and dependence responses to morphine [35,37,46,53]. binding incubation period with [ H]DPCPX or3Although the first m-knockout animals showed no differ- [ H]CGS21680 the slides were washed three times for 5

ences in pain sensitivity in the tail flick and hot plate min with ice-cold rinse buffer (170 mM Tris–HCl, pH 7.4thermal tests [37], recent studies showed hyperalgesia in at 0 8C) followed by a 5 min rinse in ice cold water. For

3thermal tests and hypoalgesia in visceral (acetic acid [ H]NBTI binding the slides were washed three times for 5writhing) and inflammatory pain tests but no alteration in min in 50 mM Tris–HCl ice cold rinse buffer (pH 7.4 at

3paw pressure tests [17,38,41,52]. 0 8C). The slides were apposed to [ H]Hyperfilm (Amer-sham) for a period of 3 weeks (A and A receptors) or 41 2A

weeks (NBTI-sensitive transporters) for brain and 5 weeks(A receptors and NBTI-sensitive transporters) or 201

2 . Materials and methods weeks (A receptors) for spinal cord. Films were de-2A

veloped using 50% Kodak D19 developer. m-Opioid3 2 4 52 .1. Generation of knockout mice receptor binding with [ H]D-Ala -MePhe -Gly-ol en-

kephalin (DAMGO), as described in detail by Kitchen etThe experimental methodology for the generation of al. [31], was also carried out in order to confirm the

mice deficient in the m-opioid receptor gene has been genotype of m-opioid receptor knockout mice.

70 A. Bailey et al. / Brain Research 943 (2002) 68 –79

2 .3. Quantitative analysis and statistical procedures ceptor knockout mice brains and spinal cords was identicalto wild type (Figs. 1 and 2). Moreover, the levels of

3Quantitative analysis of brain sections was carried out as [ H]DPCPX specific binding of the regions of cervicaldetailed previously [31] using an MCID image analyser spinal cord analysed were very similar to those of the(Imaging Research, Canada). Brain structures were iden- respective regions of the thoracic and sacral lumbar spinaltified by reference to the mouse atlas of Franklin and cord analysed (Table 2), suggesting that the pattern ofPaxinos [14] and spinal cord structures were identified by distribution of A receptors in the spinal cord is homoge-1

using the rat atlas of Paxinos and Watson [39]. Measure- neous along its length.ments for quantitative analysis of spinal cords were taken Two-way ANOVA demonstrated a significant quantita-

3from both right and left side for each region, therefore tive difference (P,0.01) in the levels of [ H]DPCPXrepresenting a duplicate determination apart from lamina X binding between genotypes in the brain (Table 1). Al-and white matter where only one measurement was taken. though the median change for all regions was only 4–5%,All anatomical areas of the spinal cord were analysed by 73% of the regions analysed in heterozygous and homo-freehand drawing. Comparison of quantitative measure- zygous knockout mice showed a downregulation in A1

ments of autoradiographic binding for each ligand in brains expression. The greatest loss of A sites (16–20%) in1

from 1 /1, 1 /2 and 2 /2 animals was carried out using homozygous brains was found in areas of high m-opioidtwo-way analysis of variance (ANOVA; for factors geno- receptor expression (nucleus accumbens shell, basolateraltype and region). To determine if there was an association amygdala). Regression analysis was carried out to de-between changes in A receptors, A receptors or NBTI termine if there was a relationship between the levels of1 2A

sensitive adenosine transporters in heterozygous or homo- expression of m-receptors in wild type brains and the%zygous mice with the level of expression of m-receptors in change in A receptors in mutant brains compared to wild1

wild type animals, a correlation analysis was carried out type, and revealed a significant correlation for the 2 /2

for all regions where m-receptors are co-expressed with A homozygous group (r50.45, n526). In spinal cords, two-1

receptors, A receptors or adenosine transporters. way ANOVA demonstrated that there were no significant2A

differences in the levels of A receptor expression between1

2 .4. Materials genotypes (P.0.05).

3 3[ H]DPCPX; 111.6 Ci /mmol, [ H]CGS21689; 42.5 Ci / 3 .3. A -receptors2A3mmol and [ H]NBTI; 30 Ci /mmol were purchased from

NEN Life Science Products (Hounslow, UK). Quantitative autoradiography of A receptors from2A3[ H]DAMGO; 56.0 Ci /mmol was purchased from Amer- coronal sections of fore, mid and hind brain of wild type

sham International (Buckinghamshire, UK). CPA, NECA, mice showed a very restricted localisation with highNBTI and adenosine deaminase type VIII were purchased binding detected in the nucleus accumbens, the caudatefrom Sigma–Aldrich (Dorset, UK). putamen and the olfactory tubercle (Table 3). The quali-

tative distribution of A receptors was identical in2A

heterozygous and homozygous mice with receptors present3 . Results in all the same regions as observed in wild type animals

(Fig. 1). Two-way ANOVA demonstrated that there were3 .1. m receptors no significant differences in the levels of A receptor2A

expression between genotypes (P.0.05). For the spinal3 3Binding of m-opioid receptors with [ H]DAMGO cord, no [ H]CGS21680 binding was observed even after

showed a loss of 50% of m-sites in animals lacking one 20 weeks film exposure time.copy of the m-opioid receptor gene and a complete absenceof m-sites in homozygous mice which confirmed the 3 .4. NBTI-sensitive adenosine transportersgenotype of the m-opioid receptor knockout mice used in

3this study. Quantitative analysis of [ H]NBTI (4.5 nM) binding inwild type brains and spinal cords revealed high binding in

3 .2. A -receptors regions such as the vertical limb of diagonal band, the1

central lateral and central medial thalamic nuclei, the3Quantitative analysis of [ H]DPCPX (3 nM) in the reuniens thalamic nucleus, the hypothalamus, the habenula,

brains and spinal cords of wild type mice revealed high the substantia nigra, the periaqueductal grey, the superficiallevels of labelling in the hippocampus, the thalamus, the grey layer of superior colliculus, the laminae I–II and X ofdentate gyrus, the subiculum, the presubiculum, in most of the spinal cord (Tables 4 and 5). The pattern of dis-the cortical regions and in the dorsal horns of the spinal tribution of NBTI-sensitive adenosine transporters in het-cord (Tables 1 and 2). The pattern of distribution of A erozygous and homozygous m-opioid receptor knockout1

receptors in heterozygous and homozygous m-opioid re- mice brains and spinal cords was identical to wild type

A. Bailey et al. / Brain Research 943 (2002) 68 –79 71

Table 1Quantitative autoradiography of A receptors in the brains of wild type (1 /1), heterozygous (1 /2) and homozygous (2 /2) m-opioid receptor knockout1

mice3Region Bregma [ H]DPCPX specific binding (fmol /mg) % Change in binding

co-ordinates(mm) Wild-type Heterozygous Homozygous (1 /2) (2 /2)

(1 /1) (1 /2) (2 /2)

Olfactory bulb 4.28Ext. plexiform layer 47.163.6 49.664.2 50.864.8 5.3 7.9Glomerular layer 48.162.9 45.164.2 49.065.5 26.2 1.9Granule layer 73.262.4 69.164.1 69.365.7 25.6 25.3Anterior olfactory nucleus 3.20 83.064.1 75.365.8 74.767.1 29.3 210.0

CortexMotor 2.46Superficial layers 14668.8 142616.2 141614.3 22.7 23.4Deep layers 16368.9 163615.2 163611.4 0.0 0.0

Rostral somatosensory 1.10Superficial layers 22369.7 208617.7 212614.2 26.7 24.9Deep layers 19568.1 192613.5 186613.3 21.5 24.6

Cingulate 1.10Superficial layers 16668.1 148614.2 150615.4 210.8 29.6Deep layers 16668.1 166615.4 167613.3 0.0 0.6

Caudal somatosensory 21.7Superficial layers 223619.0 206621.0 214617.1 27.6 24.0Deep layers 209610.5 195618.6 210610.2 26.7 0.5

Auditory 22.80Superficial layers 182610.7 180620.3 171610.4 21.1 26.0Deep layers 21668.7 208626.0 214614.8 23.7 20.9Visual 22.80

Superficial layers 203611.6 170620.5 175614.9 216.3 213.8Deep layers 218611.2 207622.6 216610.2 25.0 20.9

Reterosplenial 22.80Superficial layers 20467.1 182618.9 18966.8 210.8 27.3Deep layers 23168.0 193619.7 20569.2 216.5 211.3

Nucleus accumbens 1.34Shell 148616.1 140620.5 11969.6 25.4 219.6Core 144612.4 138615.2 132610.3 24.2 28.3

Caudate-putamen 1.10 17868.6 171615.5 175612.3 23.9 21.7Septum 0.86Medial 81.863.9 80.567.8 76.364.6 21.6 26.7Lateral dorsal 192621.4 194617.1 201619.3 1.0 4.7Lateral intermediate 163619.3 170623.6 168632.1 4.3 3.1

Vertical limb of diagonal band 0.86 69.163.3 66.165.2 63.764.5 24.3 27.8Corpus callosum 0.86 10367.1 104610.1 11065.9 1.0 6.8Ventral pallidum 0.14 14168.8 146624.9 128612.1 3.6 29.2Globus pallidus 20.10 12967.2 144614.4 139612.9 11.6 7.8Thalamus 21.70 23966.3 223619.0 219611.1 26.7 28.4

Lateroposterior nucleus 271610.6 265618.4 265613.1 22.2 22.2Ventroposterior nucleus 169611.3 167618.7 139610.2 21.2 217.8

Amygdala 21.70 82.868.7 84.2610.1 81.465.2 1.7 21.7Basolateral 169617.8 151620.3 141616.5 210.7 216.6

Hypothalamus 21.70 76.867.9 75.6610.5 69.465.6 21.6 29.6Hippocampus 22.46

Stratum oriens 315615.1 307611.5 324613.5 22.5 2.9Stratum radiatum 21667.3 186619.3 199611.8 213.9 27.9Stratum moleculare 314610.9 298620.2 290619.2 25.1 27.6

Dentate gyrus 22.46 199621.6 200620.5 208620.2 0.5 4.5Medial mammillary nucleus 22.80 12968.5 112614.8 110615.4 213.2 214.7Periaqueductal grey 23.40 11162.7 103614.9 10268.5 27.2 28.1Substantia nigra 23.40 95.264.7 98.162.8 9564.7 3.0 20.2Subiculum 23.40 24469.6 206621.4 22969.3 215.6 26.1Superficial grey layer of superior colliculus 23.40 16268.1 156614.6 143612.7 23.7 211.7Presubiculum 24.04 196610.2 196616.7 19868.2 0.0 1.0

3The mean specific binding (n55) of [ H]DPCPX (fmol /mg)6S.E.M. in brain regions of wild type (1 /1), heterozygous (1 /2) and homozygous (2 /2)m-opioid receptor knockout mice. Measures in the regions were carried out at the bregma co-ordinates taken from the mouse atlas of Franklin and Paxinos[14]. Regional determinates were made from both left and right sides of the sections, which were cut 300 mm apart. The labelling was carried out onsections from 1 /1, 1 /2 and 2 /2 mice in a completely paired protocol. Specific binding was .80% in all regions. Comparison of genotypes wasstatistically significant (P,0.01, ANOVA). The percent (%) change in binding represents the change in binding levels in 1 /2 or 2 /2 brains compared to1 /1. A minus sign indicates a percent decrease in binding levels. The overall mean and median percent changes across regions were 24.1% (1 /2),24.7% (2 /2) and 23.9 (1 /2), 24.9% (2 /2), respectively.

72 A. Bailey et al. / Brain Research 943 (2002) 68 –79

Table 2Quantitative autoradiography of A adenosine receptors in the spinal cords of wild type (1 /1), heterozygous (1 /2) and homozygous (2 /2) m-opioid1

receptor knockout mice3Region Segments [ H]DPCPX specific binding (fmol /mg) % Change in binding

Wild-type Heterozygous Homozygous (1 /2) (2 /2)(1 /1) (1 /2) (2 /2)

Cervical C1&C6Superficial layers (laminae I&II) 10969.7 107614.4 105610.4 21.8 23.7Laminae III–VI 94.867.9 92.0612.7 107.0611.5 23.0 12.9Lamina X 89.0611.7 77.667.6 91.8614.1 212.8 3.1Ventral horn (laminae VII–IX) 84.866.4 80.7610.5 91.668.1 24.8 8.0Dorsal horn (laminae I–VI) 99.868.5 99.9614.5 106610.4 0.1 6.2White matter 22.361.64 23.864.8 22.864.1 6.7 2.2

Thoracic T3&T6Superficial layers (laminae I&II) 116610.7 108612.1 117613.4 26.9 0.9Laminae III–VI 99.969.7 98.169.3 111613.2 21.8 11.1Lamina X 91.569.6 83.168.9 90.5613.8 29.2 21.1Ventral horn (laminae VII–IX) 78.267.1 76.066.8 87.6610.0 22.8 12.0Dorsal horn (laminae I–VI) 108610.3 10169.7 112612.4 26.5 3.7White matter 20.062.3 19.663.3 21.362.0 22.0 6.5

Lumbar-sacral S4Superficial layers (laminae I&II) 110611.6 101626.4 102613.9 28.2 27.3Laminae III–VI 95.367.0 96.9620.4 98.0611.9 1.7 2.8Lamina X 86.368.2 85.3628.4 85.3611.8 21.2 21.2Ventral horn (laminae VII–IX) 76.565.6 80.2614.7 84.8610.4 4.8 10.8Dorsal horn (laminae I–VI) 10469.5 101628.4 101613.6 22.9 22.9White matter 26.862.3 24.162.3 26.963.7 210.1 0.4

3The mean specific binding (n54) of [ H]DPCPX (fmol /mg)6S.E.M. in spinal cord regions of wild type (1 /1), heterozygous (1 /2) and homozygous(2 /2) m-opioid receptor knockout mice. Measures in the regions were carried out at the bregma co-ordinates taken from the rat atlas of Paxinos andWatson [39]. Regional determinates were made from both left and right sides of the sections, which were cut 300 mm apart. The labelling was carried outon sections from 1 /1, 1 /2 and 2 /2 mice in a completely paired protocol. Specific binding was .80% in all regions. Comparison of genotypes was notstatistically significant (P.0.05, ANOVA). The percent (%) change in binding represents the change in binding levels in 1 /2 or 2 /2 spinal cordscompared to 1 /1. A minus sign indicates a percent decrease in binding levels.

(Figs. 1 and 2). Two-way ANOVA demonstrated a signifi- tremely low non-specific binding. In wild-type mice thecant quantitative difference (P,0.001) in the levels of qualitative pattern of A , A receptors and NBTI-sensi-1 2A

3[ H]NBTI binding between genotypes in the brain. Thirty- tive transporters was similar to the studies that have beensix brain regions out of 46 analysed in heterozygous brains reported by other groups in rat or mouse brain and spinalshowed a downregulation compared to wild type. There cord [11,12,18–20,23,24,33,34,50,64].

3was an overall decrease in NBTI-sensitive adenosine The significant decrease in [ H]DPCPX binding intransporters binding with a median change of 11%. In mutant brains clearly suggests an interaction betweencontrast with the brain, there was no significant difference opioid and adenosine systems which is in agreement within the levels of NBTI-sensitive adenosine transporters the large body of evidence indicating the involvement ofexpression in spinal cords between genotypes (P.0.05, adenosine in a number of functional opioid effects includ-ANOVA). No significant correlation was found between ing antinociception and the expression of tolerance andlevels of expression of m-receptors in wild type brains and dependence. The nucleus accumbens shell and basolateralthe % change in the NBTI-sensitive adenosine transporter amygdala, which have very high levels of m receptorbinding in mutant mice compared to wild type (r50.18, expression in wild type animals, were the regions whichn526 for 1 /2; r50.16, n526 for 2 /2 brains). showed the greatest downregulation of A binding (16–1

20%). These decreases in binding correlated significantlywith the levels of m receptor expression in wild type

4 . Discussion animals suggesting that where m-receptors were in abun-dance there were decrements in A receptor binding1

3 3 3[ H]DPCPX, [ H]CGS21680 and [ H]NBTI were following the loss of these m receptors, implying thechosen to label selectively A receptors, A receptors and existence of a m–A receptor functional interaction in the1 2A 1

NBTI-sensitive adenosine transporters not only because of brain. This supports the assertion that has been made bytheir receptor or transporter selectivity (140–150,000-fold Aley and Levine [3] that there is a m, a , A physical2 1

[23,36,63]) but also because all three ligands have ex- receptor complex mediating antinociception in the

A. Bailey et al. / Brain Research 943 (2002) 68 –79 73

Fig. 1. Computer enhanced autoradiograms of coronal brain sections from wild type (1 /1), heterozygous (1 /2) and homozygous (2 /2) m-opioidreceptor knockout mice. The sections shown are from the level of the caudate (bregma 1.10 mm) and the hippocampus (bregma 21.70 mm) for A receptor1

and NBTI-sensitive adenosine transporter binding and at the level of the caudate (bregma 1.10 mm) and the globus pallidus (bregma 20.22 mm) for A2A3 3receptor binding. A receptors were labelled with [ H]DPCPX (3 nM), A receptors with [ H]CGS21680 (10 nM) and NBTI-sensitive adenosine1 2A

3transporters with [ H]NBTI (4.5 nM). The colour bar shows pseudo-colour interpretation of relative density of black and white film image calibrated infmol /mg tissue. Sections from 1 /1, 1 /2 and 2 /2 brains were processed in parallel.

periphery. Furthermore, it has been suggested that m–A highly expressed [31] and d-receptors have also been1

interactions could take place at the level of the second implicated in regulation of adenosine release [5].messenger (G or G proteins) as both receptors share a A reduction of adenosine release which might resulti o

common second messenger pathway in the induction of from the lack of m-opioid receptor stimulation in 2 /2

tolerance and withdrawal [3,45]. It is also worthy of note mice brains seems unlikely to be the mechanism by whichthat d-opioid receptors are down-regulated in m-knockout A receptor number is decreased in 2 /2 brains. A1

mice, especially in regions where m-receptors are normally reduction in adenosine release would most probably lead to

74 A. Bailey et al. / Brain Research 943 (2002) 68 –79

Fig. 2. Computer enhanced autoradiograms of coronal cervical (rows a and d), thoracic (rows b and e) and sacral (rows c and f) spinal cord sections fromwild type (1 /1), heterozygous (1 /2) and homozygous (2 /2) m-opioid receptor knockout mice. The sections taken were C6 segment for cervical, T3 for

3thoracic and S4 for sacral spinal cord according to the rat atlas of Paxinos and Watson [39]. A receptors were labelled with [ H]DPCPX (3 nM) and13NBTI-sensitive adenosine transporters with [ H]NBTI (4.5 nM). The colour bar shows pseudo-colour interpretation of relative density of black and white

film image calibrated in fmol /mg tissue. Sections from 1 /1, 1 /2 and 2 /2 spinal cords were processed in parallel.

an upregulation of A receptors, as opposed to the more likely that the lack of stimulation of m-opioid1

observed downregulation. In support of this, it has been receptors in the knockout mice is the direct cause of theshown that adenosine A receptors were downregulated changes in A receptors, and this is supported by the1 1

following prolonged incubation of adipocytes with an A opposite upregulation in A receptors which has been1 1

receptor agonist phenylisopropyl-adenosine (PIA), whereas reported to occur after chronic morphine treatment [2,27].an upregulation of brain A receptors was observed in As far as changes in A receptor numbers in 1 /2 and1 2A

chronically caffeine-treated animals [4,15,22,66]. It is 2 /2 mice brains are concerned, no significant overall

A. Bailey et al. / Brain Research 943 (2002) 68 –79 75

Table 3Quantitative autoradiography of A receptors in the brains of wild type (1 /1), heterozygous (1 /2) and homozygous (2 /2) m-opioid receptor knockout2A

mice3Region Bregma [ H]CGS21680 specific binding (fmol /mg) % Change in binding

co-ordinates(mm) Wild-type Heterozygous Homozygous (1 /2) (2 /2)

(1 /1) (1 /2) (2 /2)

Nucleus accumbens 1.34Shell 209615.0 191617.2 182618.9 28.6 212.9Core 18268.1 175615.6 171615.2 23.8 26.0

Caudate-putamen 1.10 296618.5 300619.5 294610.2 1.4 20.7Olfactory tubercle 1.10 274616.2 266621.4 252617.1 22.9 28.0Globus pallidus 20.10 87.768.1 93.563.9 98.065.7 6.6 11.7

3The mean specific binding (n55) of [ H]CGS21680 (fmol /mg)6S.E.M. in brain regions of wild type (1 /1), heterozygous (1 /2) and homozygous(2 /2) m-opioid receptor knockout mice. Measures in the regions were carried out at the bregma co-ordinates taken from the atlas of Franklin and Paxinos[14]. Regional determinates were made from both left and right sides of the sections, which were cut 300 mm apart. The labelling was carried out onsections from 1 /1, 1 /2 and 2 /2 mice in a completely paired protocol. Specific binding was .95% in all regions. Comparison of genotypes was notstatistically significant (P.0.05, ANOVA). The percent (%) change in binding represents the change in binding levels in 1 /2 or 2 /2 brains compared to1 /1. A minus sign indicates a percent decrease in binding levels. The overall mean and median percent changes across regions was 21.5% (1 /2),23.2% (2 /2) and 22.9% (1 /2), 26.0% (2 /2), respectively.

changes were observed which also accords with a study in affinity. Although the use of a single concentration ofthat failed to find changes in A receptor binding in radioligand does not allow discrimination between a2A

brains of chronically morphine treated mice [27]. Although change in receptor number or a change in receptor affinity,one study in rats has suggested an upregulation of A homogenate binding carried out by several groups in2A

receptors in chronically morphine-treated rats [65], the chronically morphine-treated mice and rats did not find anycurrent study suggests that A -m receptor interactions are change in affinity for either A receptors or NBTI sensitive2A 1

not relevant in the mouse. Moreover, it would be worth- adenosine transporters whilst the B had changed sig-max

while mentioning that no binding of A receptors was nificantly [26,59,60]. In addition, homogenate binding2A

observed in the spinal cord of wild type or knockout mice. carried out by us in d knockout mice [13] found no changeThis is in agreement with a study that showed no signifi- in affinity for either m and k receptor ligands. We found thecant expression of the A R gene in the rat spinal cord same was true for m and k knockouts [37,49]. As a result,2A

[25]. it is most likely that the changes observed in binding at aThe overall significant downregulation of NBTI sensi- single ligand concentration do reflect changes in receptor

tive adenosine transporters in brains of m-knockout mice is density rather than receptor affinity. Whether the smallconsistent with the study by Kaplan and Leite-Morris [26] changes in receptor and transporter expression translates tothat showed upregulation of adenosine transporter binding biologically relevant effects remains to be determined, butsites in the striatum and hypothalamus of opiate tolerant it should be noted that in heterozygous m-knockout micemice. The decrease in NBTI binding could be due to the the 50% loss of receptors correlates with a sixfold loss ofreduction of m-receptors (G , G coupled) in mutant brains analgesia [51] and we have recently shown significanti o

which would lead to an increased activation of adenylate changes in opioid analgesia in A knockout mice which2A

cyclase (and expected increase of cyclic AMP) and an correlate with changes of receptor expression which areincreased activation of the protein kinase A pathway. less than 20% (Bailey et al., unpublished). It is also worthActivation of the cyclic AMP pathway was demonstrated noting that the effects on the A receptor and NBTI1

to result in a significant reduction of NBTI sensitive sensitive adenosine transporter are not related to geneadenosine transporter in Neuro-2A neuroblastoma and dosage and in several structures the changes are as markedchromaffin cells [47,48]. However, the lack of significant in heterozygous as well as homozygous mice. The lack ofcorrelation between levels of expression of m-receptors in gene dosage on compensatory changes in receptors has1 /1 brains and % change of NBTI-sensitive transporters been seen previously by us in ORL1 knockout mice [6]in knockout brains implies that their interaction is more and it is thus evident that loss of 50% of receptors can becomplex than this and that other factors linked to m-opioid as disruptive to the animal as complete deletion of thereceptor function could be involved in the regulation of gene.NBTI-sensitive adenosine transporters. In contrast with the brain, no significant changes in A1

The changes observed in both A receptor and NBTI- receptor and NBTI sensitive adenosine transporter numbers1

sensitive adenosine transporter binding observed in m- were observed in the spinal cord of mutant mice, sug-receptor knockout mice are more likely to be due to gesting a lack of interaction at the spinal level. That ischanges in receptor or transporter number than to changes unexpected considering the large body of evidence sug-

76 A. Bailey et al. / Brain Research 943 (2002) 68 –79

Table 4Quantitative autoradiography of the NBTI sensitive adenosine transporters in the brains of wild type (1 /1), heterozygous (1 /2) and homozygous (2 /2)m-opioid receptor knockout mice

3Region Bregma [ H]NBTI specific binding (fmol /mg) % Change in bindingco-ordinates(mm) Wild-type Heterozygous Homozygous (1 /2) (2 /2)

(1 /1) (1 /2) (2 /2)

Olfactory bulbAnterior olfactory nucleus 3.20 11668.9 116615.3 93.868.2 0 219.1

CortexMotor 2.46Superficial layers 49.165.0 51.168.0 48.163.2 4.1 22Deep layers 58.766.7 53.868.2 58.967.3 28.3 0.3

Pre-limbic 2.46 94.768.3 80.964.7 84.868.5 214.6 210.5Rostral somatosensory 1.10Superficial layers 36.665.1 40.867.3 41.162.5 11.5 12.3Deep layers 66.364.0 57.968.8 65.067.1 212.7 22

Cingulate 1.10Superficial layers 64.469.5 54.569.4 49.164.2 215.4 223.8Deep layers 65.267.2 54.067.5 46.662.9 217.2 228.5

Caudal somatosensory 21.7Superficial layers 36.864.4 38.1611.3 58.969.7 3.5 60Deep layers 62.363.8 57.269.5 73.066.0 28.2 17.2

Auditory 22.80Superficial layers 87.5610 69.265.9 90.068.8 220.9 2.9Deep layers 85.869.6 84.669.0 98.169.6 21.4 14.3

Visual 22.80Superficial layers 54.5612.7 44.368.3 41.7 218.7 223.5Deep layers 66.768.7 54.868.9 66.9612.3 217.8 0.3

Retrosplenial 22.80Superficial layers 48.267.7 36.963.4 55.764.1 223.4 15.6Deep layers 55.0614.5 31.668.9 47.368.4 242.5 214.0

Nucleus accumbens 1.34 147612.6 13166.7 13063.9 210.9 211.6Caudate-putamen 1.10 14269.2 131610.7 13064.9 27.7 28.5Septum 0.86Medial 191617.8 175619.0 173618.8 28.4 29.4Lateral 88.067.4 85.4616.3 80.068.1 23.0 29.1

Vertical limb of diagonal band 0.86 24567.9 227632.7 198611.8 27.3 219.2Globus pallidus 20.10 14664.7 14066.6 14866.1 24.1 1.4Thalamus 21.70 17967.9 15665.9 176613.7 212.8 21.7Thalamic nuclei 21.70Laterodorsal (dorsomedial) 14467.8 148612.3 174621.0 2.8 20.8Laterodorsal (ventrolateral) 130611.0 12364.3 167622 25.4 28.5Central lateral 212610.5 177615.6 16569.3 216.5 222.2Central medial 239617.8 242624.7 19269.9 1.3 219.7Ventrolateral 13069.0 96.767.0 139619.3 225.6 6.9Ventromedial 13564.4 111610.4 151618.0 217.8 11.9Ventral posterolateral 73.768.3 90.4616.0 103616.4 22.7 39.8Ventral posteromedial 97.764.9 87.766.1 115620.7 210.2 17.7Reuniens 408637.6 343638.5 336626.2 215.9 217.6Reticular 90.768.9 70.663.0 95.6614.6 222.2 5.4

Amygdala 21.70 93.766.4 94.7614.5 117613.6 1.1 24.9Hypothalamus 21.70 221618.8 187619.7 216621.1 215.4 22.3Hypothalamic nuclei 21.70Dorsomedial 263623.7 193626.3 236612.9 226.6 210.3Ventromedial 223629.8 178632.1 218621.1 220.2 22.2

Hybenula 21.70 382615.2 293621.8 354644.4 223.3 27.3Hippocampus 22.46Stratum oriens 30.562.5 32.563.2 30.465.4 6.6 20.3Stratum radiatum 23.164.1 28.267.7 30.764.1 22.1 32.9Stratum molecular 82.063.7 72.6611.6 88.364.3 211.5 7.7

Dentate gyrus 22.46 92.063.1 78.5612.5 86.668.8 214.7 25.9Medial mammillary nucleus 22.80 24866.1 240623.3 24064.0 23.2 23.2Periaqueductal grey 23.40 283611.3 254639.7 29060.9 210.2 2.5Substantia nigra 23.40 312636.4 277631.5 296623.0 211.2 25.1Superficial grey layer of superior colliculus 23.40 234625.3 15669.9 246614.7 233.3 5

3The mean specific binding (n53) of [ H]NBTI (fmol /mg)6S.E.M. in brain regions of wild type (1 /1), heterozygous (1 /2) and homozygous (2 /2)m-opioid receptor knockout mice. Measures in the regions were carried out at the bregma co-ordinates taken from the atlas of Franklin and Paxinos [14].Regional determinates were made from both left and right sides of the sections, which were cut 300 mm apart. The labelling was carried out on sectionsfrom 1 /1, 1 /2 and 2 /2 mice in a completely paired protocol. Specific binding was .70% in regions of high binding. Comparison of genotypes wasstatistically significant (P,0.001, ANOVA). The percent (%) change in binding represents the change in binding levels in 1 /2 or 2 /2 brains comparedto 1 /1. A minus sign indicates a percent decrease in binding levels. The overall mean and median percent changes across regions was 210.1% (1 /2),1.1% (2 /2) and 211.1% (1 /2), 21.8% (2 /2), respectively.

A. Bailey et al. / Brain Research 943 (2002) 68 –79 77

Table 5Quantitative autoradiography of the NBTI sensitive adenosine transporters in the spinal cords of wild type (1 /1), heterozygous (1 /2) and homozygous(2 /2) m-opioid receptor knockout mice

3Region Segments [ H]NBTI specific binding (fmol /mg) % Change in binding

Wild-type Heterozygous Homozygous (1 /2) (2 /2)(1 /1) (1 /2) (2 /2)

Cervical C1&C6Superficial layers (laminae I&II) 171610.5 193616.0 18969.9 12.9 10.5Laminae III–VI 80.564.8 85.4610.9 83.564.2 6.1 3.7Lamina X 16664.4 170616.4 168614.5 2.4 1.2Ventral horn (laminae VII–IX) 72.164.6 73.768.0 63.562.0 2.2 211.9Dorsal horn (laminae I–VI) 10567.1 12368.0 12566.8 17.1 19.0

Thoracic T3&T6Superficial layers (laminae I&II) 202614.0 182614.4 184616.6 29.9 28.9Laminae III–VI 90.065.0 88.363.3 86.167.1 21.9 24.3Lamina X 171612.2 15668.9 164611.8 28.8 24.1Ventral horn (laminae VII–IX) 76.265.9 84.065.4 77.764.9 10.2 2.0Dorsal horn (laminae I–VI) 12969.4 12267.5 120610.3 25.4 27.0

Sacral S4Superficial layers (laminae I&II) 16268.7 189610.8 182618.2 16.7 12.3Laminae III–VI 10066.8 97.764.5 92.567.4 22.3 27.5Lamina X 167610.3 176616.8 189610.6 5.4 13.1Ventral horn (laminae VII–IX) 86.367.7 86.166.0 73.063.6 20.2 215.4Dorsal horn (laminae I–VI) 12262.7 13464.5 12768.4 9.8 4.1

3The mean specific binding (n53) of [ H]NBTI (fmol /mg)6S.E.M. in spinal cord regions of wild type (1 /1), heterozygous (1 /2) and homozygous(2 /2) m-opioid receptor knockout mice. Measures in the regions were carried out at the bregma co-ordinates taken from the atlas of Paxinos and Watson[39]. Regional determinates were made from both left and right sides of the sections, which were cut 300 mm apart. The labelling was carried out onsections than 1 /1, 1 /2 and 2 /2 mice in a completely paired protocol. Specific binding was .80% in all regions and non-specific binding was low,though higher from the level of the background. Comparison of genotypes was not statistically significant (P.0.05, ANOVA). The percentage (%) changein binding represents the change in binding levels in 2 /2 or 1 /2 spinal cords compared to 1 /1. A minus sign indicates a percentage decrease inbinding levels.

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