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Quantitative distribution of the delta opioid receptor mrna in the mouse and rat CNS
Life Sciences., Vol. 56, No. 26 pp. 234523.55,1995 Copyright 0 1995 Ekevier Science Ltd Printed in the USA. AU rights rcmved 0024-3205195 s9.m + .cm
Pergamon
0024-3205(95)0022&6
QUANTITATIVE DISTRIBUTION OF THE DELTA OPIOID mRNA IN THE MOUSE AND RAT CNS
S. Jenab, B. Kest, S.O. Franklin Department
RECEPTOR
and C.E. Inturrisi
of Pharmacology, Cornell University New York, New York 10021
Medical
College,
(Received in final form March 25,1995)
SUmnKW We have used a sensitive solution hybridization assay that employs a riboprobe obtained from the mouse delta opioid receptor (DOR) coding sequence to qua&ate the relative abundance of DOR mRNA in the central nervous system (CNS) of the adult mouse and rat. In brain Poly A+ RNA extracts this riboprobe hybridized to a single 10 kb transcript from mouse and two transcripts, one of 12 and the other of 4.5 kb in size from rat. In mouse CNS the highest levels of DOR mRNA were found in the caudate-putamen at 3.3 + 0.1 (SEM) pg/ug RNA. DOR mRNA levels in the range from 2.6 to 2.1 pg/ug RNA were measured in frontal cortex, nucleus accumbens, whole brain and olfactory tubercle. Spinal cord, periaqueductal gray and hippocampus had DOR mRNA levels in the range from 1.8 to 1.5 pg/ug RNA, while medial thalamus and cerebellum had the lowest levels (0.5 pg/ug RNA). These results correlate with the reported relative distribution of DOR mRNA in mouse using an in situ hybridization technique. In rat CNS, the highest levels of DOR mRNA were measured in caudate-putamen at 2.3 + 0.1 pg equivalents/ug RNA. Whole brain, cerebral cortex, olfactory bulb and brain stem had levels in the range from 1.5 to 0.9 pg equivalents/ug RNA while the lowest DOR mRNA levels were measured at 0.5 pg equivalents/ug RNA or less in thalamus, hippocampus, substantia nigra and cerebellum. This study demonstrates the ability of solution hybridization assays to quantitate homologous (mouse) as well as similar but heterologous (rat) DOR mRNA levels.
Key Wonis: delta opioid receptor, RNA, solution hybridization, CNS regions
Three categories of opioid receptors designated as the mu, delta and kappa types transduce the pharmacodynamic effects of the opioids (l-3). Until recently (see below) the evidence for these distinct types of opioid receptors was inferred from pharmacological data obtained by the use of selective agonists and antagonists (3-6). Autoradiographic analysis has shown a distinct localization of each of the opioid receptor types in mouse and rat CNS Correspondence to: Dr. Shirzad Jenab, Pharmacology, LC524, Cornell University Medical College, 1300 York Avenue, New York, NY 10021. Tel (212) 746-6237, FAX (212) 7468835
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(1,2,7,8). The DOR is present in the highest density in mouse and rat forebrain, areas which include the frontal cortex, olfactory tubercle, nucleus accumbens and caudate-putamen (1,2,7-9). The recent cloning of a mouse DOR from NG108-15 cells (10,ll) has facilitated the cloning of the cDNAs for two other opioid receptors (12-16) and has allowed the study of the distribution of their mRNAs by in situ hybridization (9,17). Mansour et al. (9) has recently compared the distribution of DOR mRNA and DOR binding in the mouse CNS. However, using a mouse DOR probe these authors were unable to measure the distribution of DOR mRNA in rat brain. This limitation appears to be due to the lower sensitivity of the hybridization signal obtained from rat DOR mRNA and the heterologous DOR probe derived from the mouse DOR cDNA. Since our report was completed Mansour et al. (30) have used a rat DOR riboprobe and in situ hybridization to determine the distribution of DOR mRNA in rat CNS. In this study we have used a mouse DOR riboprobe to quantify, by solution hybridization, the levels of DOR mRNA from selected mouse and rat CNS areas. In mouse CNS an excellent correlation appears to exist between the relative abundance of DOR mRNA levels obtained by solution hybridization and the published in situ hybridization analysis. Furthermore, using the mouse DOR riboprobe we show a quantitative distribution for DOR mRNA in the rat CNS. Methods Male adult CD-l mice (25-35 g) were housed five to a cage in a controlled light and temperature environment with the unlimited availability of food and water. The animals were sacrificed by decapitation and the brain and spinal cord removed. The brain was cut into 1 mM thick sections using a McIlwain tissue chopper (Brinkmann Instruments, Westbury New York). The sections were transferred onto ice-cold glass slides and distinct regions of the brain were dissected by 2 or 3 mm tissue corers and homogenized in an RNA extraction buffer which consists of a denaturing solution containing guanidine isothiocyanate (18). Individual samples were pooled from 10 mice and each experiment was repeated once. The isolated brain regions were as follows: nucleus acumbens (Acb), medial thalamus (MThal from 20 mice), periaqueductal gray (PAG), olfactory tubercle (OTu), caudate-putamen (CPU), frontal cerebral cortex (FCtx), hippocampus (Hip), spinal cord (SpC, lumbar-sacral segments) cerebellum (2 whole, Cbl) and whole brain (minus cerebellum). A detailed stereotaxic mouse atlas of the cerebrum and spinal cord is not presently available. We have adapted the dissection technique for the mouse from our previously described microdissection technique for the rat (31), utilizing the detailed neuroanatomical descriptions of rat CNS neuroanatomy outlined in the atlas of Paxinos and Watson (32). Male adult Sprague-Dawley rats (125150 g) were sacrificed by decapitation and selected areas of the brain were dissected free hand and immediately homogenized in RNA extraction buffer. The tissues included the caudate-putamen (i.e., striatum) (CPU), cerebral cortex (Ctx), brain stem, (whose rostra1 and caudal boundaries were defined by the cerebellum) (BS), olfactory bulb (OB), hippocampus (Hip), thalamus (Thal), substantia nigra (SN), cerebellum (Cbl) and whole brain (minus cerebellum).
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RNA extraction Total cellular RNA was isolated by guanidine-phenol extraction and ethanol precipitation using a procedure that results in a recovery of RNA that averages 77.0 + 7.2% (SD) (18). Twenty ug of glycogen (Boehringer-Mannheim, GmbH, Germany) was used as a carrier during the RNA precipitation step. Brain poly A+ RNA was prepared as described by Sambrook et al. (19) from RNA extracts that contained 2 mg of total RNA and 0.1 mg of oligo dT cellulose (Sigma). RNA nrobes A 1058 bp PstI-Sac1 DNA fragment containing all of the coding region of the DOR cDNA was cloned into PstI and Sac1 sites of pGEM3Zf+ (Promega) (20). This DNA fragment was obtained by PCR using the DOR plasmid (10) and two primers one complementary to the DOR cDNA at positions 79 to 102, containing a PstI site, and second primer complementary to positions 1118 to 1137 that was followed by a synthetic Sac1 site. A 1074 base long 32P labelled antisense riboprobe (spec. act. = 4 x 10’ dpm/ug) was prepared using a T7 RNA polymerase transcription system. A 1082 base long unlabelled sense transcript (the calibration standard) was prepared using a SP6 RNA polymerase transcription system. A 32P 18s rRNA riboprobe (spec. act. = 1.0 X 10 7 dpmug) was prepared using a SP6 RNA polymerase transcription system and the plasmid pS/E (21). The riboprobes were purified by a CFll column as described by Franklin et al. (22) and Zhu et al. (23). Solution
hybridization
and ribonuclease
nrotection
assays
For each assay, duplicate aliquots of total RNA extracts were dried in a 1.5 ml Eppendorf microcentrifuge tube and resuspended in 30 ul of hybridization buffer (10 mM EDTA, 0.3 M NaCl, 0.5% sodium dodecylsulfate and 10 mM N-Tris[hydroxymethyl] methyl-2-amino-ethanesulfonic acid, pH 7.4) containing 150,000 dpm of the DOR riboprobe or 85,000 dpm of 18s riboprobe. The solution was covered with 2 drops of mineral oil and incubated at 75°C for 4 hours and then subjected to 40 ug/ml ribonuclease A and 2 ug/ml ribonuclease Tl for 1 hour at 30°C in 300 ul of 0.3 M NaCl, 5 mM EDTA and 10 mM Tris-HCl pH 7.4. The ribonuclease digestion was terminated with 1 ml of 5% TCA and 0.75% sodium pyrophosphate and one drop of 0.5% BSA was added to assist in the precipitation of the RNA. The solution was mixed and the TCA precipitable dpms were collected onto glass fiber paper (Brandel, Gaithersburg, MD) using a 24 place cell harvester and counted by liquid scintillation in 5 ml of Hydrofluor (National Diagnostics, Manville, NJ). Comparisons were made with standard calibration curves to quantify the DOR mRNA Total RNA concentrations were determined by a solution and total RNA levels. hybridization assay using an 18s rRNA riboprobe and a calibration curve established from rat liver total RNA standards as described by Franklin et al. (24) and Zhu et al. (23). For the ribonuclease protection assays samples of poly A+ RNA were subjected to solution hybridization and ribonuclease digestion followed by three phenol extractions and electrophoresis using a 4% denaturing polyacrylamide gel (20,22,23). Northern
blot analvsis
Samples of Poly A+ RNA were denatured in 1M glyoxalRO% dimethyl sulfoxide (v/v) for 60 minutes at 50°C fractionated by a 1.2% agarose gel and transferred to a
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nitrocellulose filter as described by Inturrisi et al., (18). To eliminate nonspecific crosshybridization, the hybridized filter was incubated with 4 @ml RNase A and 0.2uglml RNase Tl in 10 mM Tris-HCl pH 7.4, 0.3 M NaCl and 5 mM EDTA for 10 minutes followed by 3 washes of 2x SSC (0.3 M NaCl, 30 mM sodium citrate pH 7.0) at room temperature. The filter was then subjected to autoradiography with Kodak XAR-5 film at 70°C with an intensifying screen (20). Data analvsis The mean and standard error (SEM) were calculated for each replicate determination of DOR mRNA levels. Results A Northern blot analysis using the DOR riboprobe and whole brain DOR Poly A+ RNA showed a single prominent ribonuclease resistant hybridization band of 10 kb for the mouse extract (Fig. 1, lane b). Following a longer exposure an additional minor hybridization band of approximately 12 kb in size was also observed. This minor band was not further characterized. The 10 kb long transcript was clearly the predominated hybridization band and it was used to estimate the amount of DOR mRNA. The rat extract shows two ribonuclease resistant hybridization bands, a well defined band at 12 kb and a diffuse band at 4.5 kb (Fig 1, lane c). To further confirm the specificity of the DOR riboprobe used in the solution hybridization assay, an aliquot of mouse and rat Poly A+ RNA extracts corresponding to 30 pgs of DOR sense transcript (as measured by solution hybridization) were subjected to a ribonuclease protection assay (Fig. 2). The protection assay exposes the samples to the same procedure as is used in the solution hybridization assay until the final step which involves a denaturing gel electrophoresis rather than the TCA precipitation and liquid scintillation counting step (see above). Since the DOR cDNA was cloned from the mouserat hybrid NG108-15 cell line, but originates from the mouse sequences, (10,ll) a single band the size of the riboprobe (approximately 1 kb) is protected by mouse poly A+ RNA (lane h). Neither E. coli tRNA nor rat liver RNA are able to protect the riboprobe from ribonuclease (lanes e and g). Thirty pgs of the sense standard produced a single prominent band of the expected size (approximately 1 kb) (lane f). In contrast, the heterologous Poly A+ RNA from rat provided incomplete protection of the mouse riboprobe resulting in multiple bands significantly smaller than the 1 kb protected fragment obtained with mouse Poly A+ RNA (compare lane i with lane h). A representative standard calibration curve for the DOR sense standard and mRNA is shown in Fig. 3. The curve is linear from 2 to 125 pg of the 1.08 kb sense transcript. The interassay coefficient of variation averaged 9.7% for 4 consecutive assays and the intraassay coefficient of variation for 30 duplicate sample determinations averaged 2.9% (20). From the information obtained with Northern blot analysis (Fig. 1) and the ribonuclease protection assay (Fig. 2) we were able to estimate the quantitative relationship between the signal obtained with the sense standard and the corresponding amount of the DOR mRNA. A factor (9.4) that allows conversion of the amount of sense standard to DOR mRNA in mouse CNS is obtained by multiplying the ratio of the size of the DOR message in mouse (10 kb) to the sense standard (1.08 kb) by the ratio of the size of the ribonuclease protected sense standard (1.08 kb) to the protected DOR message (1.06 kb)
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(see also 23). The lowest DOR sense calibration standard is 1.95 pg (approximately 18.3 pgs of DOR mRNA). Therefore, the lower limit of sensitivity for a sample containing 120 ugs of total RNA is 0.15 pg/ug RNA. In a concurrent solution hybridization assay the signal from a mouse or rat liver RNA extract, a tissue that does not express the DOR gene is less than 0.15 pg/ug and therefore below the limit of sensitivity of the assay.
Fig. 1 A Northern blot analysis of mouse and rat brain RNA extracts. Fifty pgs of DOR sense standard (Lane a), Poly A+ RNA from mouse (lane b, 10 ug) or rat (lane c, 15 ug) brain was separated on a 1.2% agarose gel, transferred to a nitrocellulose filter hybridized with the DOR riboprobe and then subjected to ribonuclease treatment. A single ribonuclease resistant hybridization band of 10 kb in length was detected in the mouse extract (lane b) while the rat extract contained a prominent 12 kb band and a diffuse 4.5 kb band (lane c). The film was exposed at -70” for 20 hrs (lanes a and b) and 36 hrs to visualize lane c.
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Fig. 2 Ribonuclease protection analysis of mouse and rat DOR mRNA. Following solution hybridization and ribonuclease (RNase) digestion the samples were subjected to a 4% denaturing polyacrylamide gel electrophoresis. Lanes a and b are the size markers. Lane d is the DOR riboprobe not subjected to solution hybridization. Lane c and lanes e through i contain samples that were carried through the solution hybridization and recovery steps. The sample in lane c is the riboprobe with the RNase treatment omitted, while lane e shows that E. coli t-RNA doses not protect the riboprobe from RNase treatment. Lane f shows the protection resulting from the DOR sense transcript (30 pg), lane g is rat liver RNA (100 ug), which failed to protect the riboprobe, lane h is mouse Poly A+ RNA (6 ug, equivalent to 30 pg of the DOR sense transcript), lane i is rat Poly A+ RNA (15 ug, equivalent to 30 pg of the DOR sense transcript). The arrow (lane i) indicates the largest fragment (approx. 0.56 kb) protected in rat Poly A+ RNA by the mouse derived DOR riboprobe. Lane i is from a separate but equivalent protection assay.
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I”
0
50
25 Delta
L 0
75
Transcript I
125
150
(pg) I
470
predicted
100
2349
940
f
1410
DOR mRNA (pg) Fig. 3
A standard calibration curve obtained by hybridization of the DOR riboprobe to sense standards. The calibration standards, which contained 5 ug of E. coli tRNA carrier and a known amount of DOR sense transcript, were hybridized to the DOR riboprobe and assayed using the solution hybridizationRNase protection-TCA precipitation protocol described in Materials and Methods. The ordinate shows the hybridization signal, i.e., the TCA-precipitable dpm minus background dpm (less than 1% of the total TCA-precipitable dpm of the incubated riboprobe). The abscissa indicates the pgs of DOR sense transcript present in a given calibration standard, while the lower axis shows the amounts of 10 kb DOR mRNA expected to yield the same hybridization signal as a particular calibration standard. The points are the average of two determinations and the solid line is the result of linear regression analysis. Fig. 4 shows the steady-state DOR mRNA levels in selected regions of adult mouse CNS in pg/ug RNA (SEM). The highest levels of DOR mRNA were found in caudateputarnen; CPU at 3.3 (0.1) and frontal cortex; FCtx at 2.6 (O.l), nucleus accumbens; Acb at 2.4 (0.1) and olfactory tubercle; OTu at 2.1 (0.1). DOR mRNA levels in pg/ug RNA (SEM) were for spinal cord; SpC at 1.8 (0.2), periaqueductal gray; PAG at 1.5 (0.1) and hippocampus; Hip at 1.6 (0.2). The lowest levels of DOR mRNA were measured in medial tbalamus; MThal at 0.6 (0.1) and cerebellum; Cbl at 0.6 (0.1). Mouse brain (without cerebellum) contained 2.2 (0.3) pg/ug RNA of DOR mRNA.
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Fig. 5 shows the corresponding distribution of DOR mRNA in the rat CNS in pg equivalents/ug RNA (SEM). The highest levels of DOR mRNA were measured in caudateputamen; CPU at 2.3 (0.1) while cerebral cortex; Ctx at 1.3 (O.l), brain stem; BS at 1.1 (0.1) and olfactory bulb; OB at 0.9 (0.1) contained lower levels. Hippocampus; Hip at 0.4 (O.l), cerebellum; Cbl at 0.4 (O.l), substantia nigra; SN at 0.3 (0.2) and thalamus; Thal at 0.5 (0.1) showed the lowest levels of the message. Rat brain (without cerebellum) contained 1.5 (. 18) DOR mRNA pg equivalents/ug RNA.
3.5 r
MOUSE
3.0 1 2.0 1.5 1.0 0.5 0.0 _1 Brain FCtx OTu Acb CPU Hip MThal PAG Cbl SpC
Fig. 4 The distribution of DOR mRNA in the mouse CNS. The solution hybridization assay was used to quantitate DOR mRNA distribution in whole brain without cerebellum (Brain), frontal cortex (FCtx), olfactory tubercle (OTu), nucleus accumbens (Acb), caudate-putamen (CPU), hippocampus (Hip), medial thalamus (MThal), periaqueductal gray (PAG), cerebellum (Cbl) and spinal cord (lumbar-sacral segments, SpC). Each value is the mean +S.E.M. for two separate experiments.
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2.5 -
0.0
I-
RAT
Brain Ctx OB
CPU
Hip Thai
SN
Cbl
1. BS
Fig. 5 The distribution of DOR mRNA in the rat CNS. The solution hybridization assay was used to qua&ate DOR mRNA distribution in whole brain without cerebellum (Brain), cerebral cortex (Ctx), olfactory bulb (OB), caudate-putamen (CPU), hippocampus (Hip), thalamus (Thal), substantia nigra (SN), brain stem (BS) and cerebellum (Cbl). Each value is the mean +S.E.M. for two separate experiments. Discussion Based on binding studies DOR are the most abundant of the three opioid receptor types (8). Expressed as a relative proportion, they represent 50% or more of opioid receptors in mouse and rat brain tissues (8). We have used a facile solution hybridization assay to quantitate the distribution of the DOR mR_NA in both mouse and rat CNS. Our assay is more sensitive than Northern blot analysis since up to 120 ug of target RNA can be used for each determination (20,23). Furthermore, by constructing a calibration curve from sense standards the assay is more quantitative than the usual ribonuclease protection assay. The 18s rRNA assay which measures total cellular RNA allows each sample to be normalized for the recovery of RNA and for the value of DOR mRNA to be expressed in pg (or pg equivalents) per ug of RNA. In Northern blot analysis (Fig. 1) our riboprobe hybridized to a single 10 kb transcript of DOR mRNA, which allowed the signal obtained with solution hybridization to be converted from the amount of sense standard to the corresponding amount of a 10 kb mRNA (Fig. 3). Interestingly, the single hybridization band found in mouse brain poly A+ RNA corresponds to the largest of the six DOR transcripts found in NG108-15 cells (25).
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The DOR cDNA was isolated from this mouse-rat hybrid cell line (10,ll) but appears to originate from the mouse sequences (11). In NG108-15 cell extracts this transcript represents about 36% of the total band intensity signal (20). It is not known to what degree each of the six DOR transcripts from NGlOS-15 cells are translated to yield receptor protein. However, these results suggest that the largest of the DOR transcripts, common to both NG108-15 cells and mouse brain may be of particular interest for further study. In the Northern blot analysis of rat brain poly A+ RNA extracts, two hybridization bands remained after ribonuclease treatment, a prominent 12 kb and a more diffuse 4.5 kb band. The presence of multiple transcripts in rat brain Poly A+ RNA extracts is consistent with three previous reports (3,10,15) but not with that of Abood et al. (12) who found only a single 6 kb band in rat brain RNA extracts. To confirm the specificity of our DOR riboprobe we used a ribonuclease protection assay (Fig. 2). As predicted from the relative lengths and sequences of the mouse DOR mRNA, the sense transcript and the riboprobe, the ribonuclease resistant duplex formed between the riboprobe and either the mouse DOR mRNA or the sense transcript resulted in a major band of approximately 1 kb. A single major band corresponding to a resistant duplex of the predicted length indicates that the signal obtained by solution hybridization is due to the mouse DOR mRNA present in the RNA extract and not to other RNAs that may have considerable homology to the cloned delta receptor (e.g., mu or kappa opioid receptor mRNAs) (10-16). Furthermore, using the sequence analysis software package (Genetics Computer Group, Inc., Madison, WI), we have determined that the longest stretch of perfect homology between the mouse DOR riboprobe and the mouse kappa receptor cDNA (16), rat kappa receptor cDNA (14) or rat mu receptor cDNA (13,lS) are respectively, 23, 23 and 32 bases in length. Therefore, it is very unlikely that sequences from either the mouse kappa, rat kappa or mu receptor mRNAs would contribute to the RNase protected-TCA precipitated counts. It is important to note that our RNase assay conditions result in complete hydrolysis of unprotected riboprobe and that “other” RNAs do not yield the same major protected band (Fig. 2 lanes e, g and i). In addition to a protected band of the expected size (Fig. 2 lanes f and h) an array of bands that are smaller in size were also noted. These bands result in part from thermal degradation of the riboprobe (compare Fig. 2 lanes c and d) and from the dynamics of the hybridization of RNAs. The hybrids formed between an mRNA or the sense transcript and the riboprobe do not remain as a duplex all of the time, rather they separate or “breathe” allowing RNase to attack even complementary strands, yielding some of the fragment bands shown in Fig. 2. This occurs in all protection assays where RNase is in excess (20, 23, 29). However, our solution hybridization assay compares the dpm obtained from all of the protected fragments that are trapped on the filter when the mouse DOR sense transcript is exposed to RNase with the array produced from the mouse DOR mRNA. Since both mouse DOR sense transcript and mouse DOR mRNA are exposed to the same conditions, then, as shown in Fig. 2, they yield the same array of bands. As expected, rat Poly A+ RNA extracts only partially protected the mouse DOR riboprobe as reflected in the multiple bands of 0.5 kb or less detected in a ribonuclease protection assay (Fig. 2 lane i). However, this degree of protection allowed the use of the mouse DOR riboprobe to estimate the relative amounts of the rat DOR mRNA levels (Fig. 5 and see below). Thus, solution hybridization allows a comparison of the relative abundance of DOR transcripts between tissues and/or species. For example, the NGlOS-15 cell line, which is the source of the original DOR cDNA clones (10,l l), contains six DOR transcripts that yield a mean value of 0.27 pg of DOR sense transcriptiug RNA (20). This value is very
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similar to the mean values of 0.23 pg of DOR sense transcript/ug RNA (equivalent to 2.2 pg/ug RNA of a 10 kb DOR mRNA) found in mouse brain RNA extracts and 0.16 pg of DOR sense transcript/ug RNA (equivalent to 1.5 pg of DOR mRNA/ug RNA) found in rat brain RNA extracts. Furthermore, the solution hybridization assay can be used to complement and extend mRNA distribution maps obtained by in situ hybridization (30). In agreement with Mansour et al., (9) we found the highest levels of DOR mRNA in mouse telencephalic structures of the limbic system including caudate-putamen, frontal cortex, olfactory tubucle, nucleus accumbens and hippocampus (Fig. 4). An excellent correlation is also obtained when the distribution of DOR binding sites in the limbic system, as measured by autoradiography (7-9), is compared with the distribution DOR mRNA measured by in situ hybridization (9) or solution hybridization (this report). This colocalization of DOR mRNA and receptors in the limbic system may permit regulation of the endogenous opioid system by activation of autoreceptors and/or via local opioidergic circuits and may be reflected in the ability of DOR selective opioids to elicit seizures (26,27) and to facilitate reward behavior (28). DOR mRNA, as expected from the results of Mansour et al., (9), was measured in the mouse spinal cord, hippocampus and whole brain (Fig. 4). However, in contrast to report of Mansour et al., (9) we were able to measure DOR mRNA in periaqueductal gray (PAG) and cerebellum. While these differences may result from the different but partially overlapping probes used in our study compared to that of Mansour et al., (9), it is more likely that our ability to increase the amount of target RNA in the solution hybridization assay accounts for the greater sensitivity (see below). In this regard it must be emphasized that our assays include a comparable amount of an RNA extract from a tissue, e.g., (mouse or rat liver) that does not express the DOR and a complete standard calibration curve so that the lower limit of sensitivity can be accurately defined for each assay. Autoradiographic studies (7) have measured DOR binding in the mouse PAG so that our results (Fig. 4) support colocalization of DOR mRNA and receptor in this mesencephalic area involved in the processing of nociceptive information (7). We attempted to qua&ate DOR mRNA levels in selected rat CNS regions with the mouse DOR riboprobe. When there is a high degree of homology in the mRNA sequences from the two species, the solution hybridization procedure can be used to measure relative mRNA levels using a heterologous probe (22,29). In ribonuclease protection assays the mouse riboprobe protected multiple bands from rat brain extracts indicating the expected sequence variation between the two species (Fig. 2). The mouse riboprobe is not completely protected by rat DOR mRNA so that our assay yields values for the rat DOR mRNA which are underestimates of the true amount. Since it is not possible to determine the exact pg amounts, the rat levels are expressed as pg equivalents which allows a relative comparison of the signal obtained from different rat CNS areas (22,29). With Northern blot analysis the mouse riboprobe hybridized to transcripts from rat brain extracts that are 12 and 4.5 kb in length and clearly a different size than the mouse DOR mRNA (Fig. 2). Since the Northern blot analysis utilized a ribonuclease treatment, the signal from rat brain which remained, represents hybridization of the rat DOR sequences with the mouse DOR riboprobe. As with the mouse, we selected for analysis rat CNS regions that have been shown by autoradiography (1,2) and in situ hybridization (30) to contain a relatively high (e.g.,
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caudate-putamen and olfactory bulb) or low (e.g., thalamus and cerebellum) density of DOR and/or DOR mRNA. As with the distribution of the mouse DOR mRNA we found that rat DOR mRNA levels are 3 to 8 fold higher in telencephalic regions than in the other brain regions we sampled (Fig. 5). These results, although limited, suggest that like the mouse DOR mRNA, a close correspondence exists between the density of DOR receptors and DOR mRNA expression in the rat CNS. Acknowledgements Supported in part by NIDA Grant DA01457 (CEI), NIDA Training Grant DA07274 (SJ). CEI is a recipient of a Research Scientist Award from NIDA (DAO0198). BK is an Aaron Diamond Foundation Fellow and this work was supported in part by The Aaron Diamond Foundation.
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S. JENAB and C.E. INTURRISI, Mol. Brain Res. 22 95-102 (1994). J.M. ERICKSON, C.L. RUSHFORD, D.J. DORNEY, G.N. WILSON and T.D. SCHMICKEL, Gene 16 l-9 (1981). S.O. FRANKLIN, Y.S. ZHU, B.C. YOBURN and C.E. INTURRISI, Mol. Pharmacol. 40 515-522 (1991). Y.S. ZHU, A.D. BRANCH, H.D. ROBERTSON, T.H. HUANG, S.O. FRANKLIN and C.E. INTURRISI, Mol. Brain Res. 12 173-180 (1992). S.O. FRANKLIN, B.C. YOBURN, Y.S. ZHU, A.D. BRANCH, H.D. ROBERTSON and C.E. INTURRISI, Mol. Brain Res. a 241-250 (1991). S. JENAB and C.E. INTURRISI, Regul. Pept. 54 135-136 (1994). D.P. CAIN, F. BOON and M.E. CORCORAN, Brain Res. 517 236-244 (1990). S.D. COMER, E.M. HOENICKE, A.I. SABLE, R.W. McNUTT, K.J. CHANG, B.R. DeCOSTA, H.I. MOSBERG and J.H. WOODS, J. Pharm. Exp. Ther. 267 888-895 (1993). L. STEIN and J.D. BELLUZZI, Federation Proc. 28 2468-2472 (1979). S.O. FRANKLIN, K. ELLIOTT, Y.S. ZHU, C. WAHLESTEDT and C.E. INTURRISI, Mol. Brain Res. 19 93-100 (1993). A. MANSOUR, C.A. FOX, S. BURKE, F. MENG, R.C. THOMPSON, H. AKIL and S.J. WATSON, J. Comparative Neurol., 350 412-438 (1994). M. BRODSKY, K. ELLIOTT, A. HYNANSKY, S. JENAB and C.E. INTURRISI, NeuroReport, in press, (1995). G. PAXINOS and C. WATSON, The Rat Brain in Stereotaxic Coordinates, Academic Press, CA (1986).
Pergamon
0024-3205(95)0022&6
QUANTITATIVE DISTRIBUTION OF THE DELTA OPIOID mRNA IN THE MOUSE AND RAT CNS
S. Jenab, B. Kest, S.O. Franklin Department
RECEPTOR
and C.E. Inturrisi
of Pharmacology, Cornell University New York, New York 10021
Medical
College,
(Received in final form March 25,1995)
SUmnKW We have used a sensitive solution hybridization assay that employs a riboprobe obtained from the mouse delta opioid receptor (DOR) coding sequence to qua&ate the relative abundance of DOR mRNA in the central nervous system (CNS) of the adult mouse and rat. In brain Poly A+ RNA extracts this riboprobe hybridized to a single 10 kb transcript from mouse and two transcripts, one of 12 and the other of 4.5 kb in size from rat. In mouse CNS the highest levels of DOR mRNA were found in the caudate-putamen at 3.3 + 0.1 (SEM) pg/ug RNA. DOR mRNA levels in the range from 2.6 to 2.1 pg/ug RNA were measured in frontal cortex, nucleus accumbens, whole brain and olfactory tubercle. Spinal cord, periaqueductal gray and hippocampus had DOR mRNA levels in the range from 1.8 to 1.5 pg/ug RNA, while medial thalamus and cerebellum had the lowest levels (0.5 pg/ug RNA). These results correlate with the reported relative distribution of DOR mRNA in mouse using an in situ hybridization technique. In rat CNS, the highest levels of DOR mRNA were measured in caudate-putamen at 2.3 + 0.1 pg equivalents/ug RNA. Whole brain, cerebral cortex, olfactory bulb and brain stem had levels in the range from 1.5 to 0.9 pg equivalents/ug RNA while the lowest DOR mRNA levels were measured at 0.5 pg equivalents/ug RNA or less in thalamus, hippocampus, substantia nigra and cerebellum. This study demonstrates the ability of solution hybridization assays to quantitate homologous (mouse) as well as similar but heterologous (rat) DOR mRNA levels.
Key Wonis: delta opioid receptor, RNA, solution hybridization, CNS regions
Three categories of opioid receptors designated as the mu, delta and kappa types transduce the pharmacodynamic effects of the opioids (l-3). Until recently (see below) the evidence for these distinct types of opioid receptors was inferred from pharmacological data obtained by the use of selective agonists and antagonists (3-6). Autoradiographic analysis has shown a distinct localization of each of the opioid receptor types in mouse and rat CNS Correspondence to: Dr. Shirzad Jenab, Pharmacology, LC524, Cornell University Medical College, 1300 York Avenue, New York, NY 10021. Tel (212) 746-6237, FAX (212) 7468835
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(1,2,7,8). The DOR is present in the highest density in mouse and rat forebrain, areas which include the frontal cortex, olfactory tubercle, nucleus accumbens and caudate-putamen (1,2,7-9). The recent cloning of a mouse DOR from NG108-15 cells (10,ll) has facilitated the cloning of the cDNAs for two other opioid receptors (12-16) and has allowed the study of the distribution of their mRNAs by in situ hybridization (9,17). Mansour et al. (9) has recently compared the distribution of DOR mRNA and DOR binding in the mouse CNS. However, using a mouse DOR probe these authors were unable to measure the distribution of DOR mRNA in rat brain. This limitation appears to be due to the lower sensitivity of the hybridization signal obtained from rat DOR mRNA and the heterologous DOR probe derived from the mouse DOR cDNA. Since our report was completed Mansour et al. (30) have used a rat DOR riboprobe and in situ hybridization to determine the distribution of DOR mRNA in rat CNS. In this study we have used a mouse DOR riboprobe to quantify, by solution hybridization, the levels of DOR mRNA from selected mouse and rat CNS areas. In mouse CNS an excellent correlation appears to exist between the relative abundance of DOR mRNA levels obtained by solution hybridization and the published in situ hybridization analysis. Furthermore, using the mouse DOR riboprobe we show a quantitative distribution for DOR mRNA in the rat CNS. Methods Male adult CD-l mice (25-35 g) were housed five to a cage in a controlled light and temperature environment with the unlimited availability of food and water. The animals were sacrificed by decapitation and the brain and spinal cord removed. The brain was cut into 1 mM thick sections using a McIlwain tissue chopper (Brinkmann Instruments, Westbury New York). The sections were transferred onto ice-cold glass slides and distinct regions of the brain were dissected by 2 or 3 mm tissue corers and homogenized in an RNA extraction buffer which consists of a denaturing solution containing guanidine isothiocyanate (18). Individual samples were pooled from 10 mice and each experiment was repeated once. The isolated brain regions were as follows: nucleus acumbens (Acb), medial thalamus (MThal from 20 mice), periaqueductal gray (PAG), olfactory tubercle (OTu), caudate-putamen (CPU), frontal cerebral cortex (FCtx), hippocampus (Hip), spinal cord (SpC, lumbar-sacral segments) cerebellum (2 whole, Cbl) and whole brain (minus cerebellum). A detailed stereotaxic mouse atlas of the cerebrum and spinal cord is not presently available. We have adapted the dissection technique for the mouse from our previously described microdissection technique for the rat (31), utilizing the detailed neuroanatomical descriptions of rat CNS neuroanatomy outlined in the atlas of Paxinos and Watson (32). Male adult Sprague-Dawley rats (125150 g) were sacrificed by decapitation and selected areas of the brain were dissected free hand and immediately homogenized in RNA extraction buffer. The tissues included the caudate-putamen (i.e., striatum) (CPU), cerebral cortex (Ctx), brain stem, (whose rostra1 and caudal boundaries were defined by the cerebellum) (BS), olfactory bulb (OB), hippocampus (Hip), thalamus (Thal), substantia nigra (SN), cerebellum (Cbl) and whole brain (minus cerebellum).
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RNA extraction Total cellular RNA was isolated by guanidine-phenol extraction and ethanol precipitation using a procedure that results in a recovery of RNA that averages 77.0 + 7.2% (SD) (18). Twenty ug of glycogen (Boehringer-Mannheim, GmbH, Germany) was used as a carrier during the RNA precipitation step. Brain poly A+ RNA was prepared as described by Sambrook et al. (19) from RNA extracts that contained 2 mg of total RNA and 0.1 mg of oligo dT cellulose (Sigma). RNA nrobes A 1058 bp PstI-Sac1 DNA fragment containing all of the coding region of the DOR cDNA was cloned into PstI and Sac1 sites of pGEM3Zf+ (Promega) (20). This DNA fragment was obtained by PCR using the DOR plasmid (10) and two primers one complementary to the DOR cDNA at positions 79 to 102, containing a PstI site, and second primer complementary to positions 1118 to 1137 that was followed by a synthetic Sac1 site. A 1074 base long 32P labelled antisense riboprobe (spec. act. = 4 x 10’ dpm/ug) was prepared using a T7 RNA polymerase transcription system. A 1082 base long unlabelled sense transcript (the calibration standard) was prepared using a SP6 RNA polymerase transcription system. A 32P 18s rRNA riboprobe (spec. act. = 1.0 X 10 7 dpmug) was prepared using a SP6 RNA polymerase transcription system and the plasmid pS/E (21). The riboprobes were purified by a CFll column as described by Franklin et al. (22) and Zhu et al. (23). Solution
hybridization
and ribonuclease
nrotection
assays
For each assay, duplicate aliquots of total RNA extracts were dried in a 1.5 ml Eppendorf microcentrifuge tube and resuspended in 30 ul of hybridization buffer (10 mM EDTA, 0.3 M NaCl, 0.5% sodium dodecylsulfate and 10 mM N-Tris[hydroxymethyl] methyl-2-amino-ethanesulfonic acid, pH 7.4) containing 150,000 dpm of the DOR riboprobe or 85,000 dpm of 18s riboprobe. The solution was covered with 2 drops of mineral oil and incubated at 75°C for 4 hours and then subjected to 40 ug/ml ribonuclease A and 2 ug/ml ribonuclease Tl for 1 hour at 30°C in 300 ul of 0.3 M NaCl, 5 mM EDTA and 10 mM Tris-HCl pH 7.4. The ribonuclease digestion was terminated with 1 ml of 5% TCA and 0.75% sodium pyrophosphate and one drop of 0.5% BSA was added to assist in the precipitation of the RNA. The solution was mixed and the TCA precipitable dpms were collected onto glass fiber paper (Brandel, Gaithersburg, MD) using a 24 place cell harvester and counted by liquid scintillation in 5 ml of Hydrofluor (National Diagnostics, Manville, NJ). Comparisons were made with standard calibration curves to quantify the DOR mRNA Total RNA concentrations were determined by a solution and total RNA levels. hybridization assay using an 18s rRNA riboprobe and a calibration curve established from rat liver total RNA standards as described by Franklin et al. (24) and Zhu et al. (23). For the ribonuclease protection assays samples of poly A+ RNA were subjected to solution hybridization and ribonuclease digestion followed by three phenol extractions and electrophoresis using a 4% denaturing polyacrylamide gel (20,22,23). Northern
blot analvsis
Samples of Poly A+ RNA were denatured in 1M glyoxalRO% dimethyl sulfoxide (v/v) for 60 minutes at 50°C fractionated by a 1.2% agarose gel and transferred to a
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nitrocellulose filter as described by Inturrisi et al., (18). To eliminate nonspecific crosshybridization, the hybridized filter was incubated with 4 @ml RNase A and 0.2uglml RNase Tl in 10 mM Tris-HCl pH 7.4, 0.3 M NaCl and 5 mM EDTA for 10 minutes followed by 3 washes of 2x SSC (0.3 M NaCl, 30 mM sodium citrate pH 7.0) at room temperature. The filter was then subjected to autoradiography with Kodak XAR-5 film at 70°C with an intensifying screen (20). Data analvsis The mean and standard error (SEM) were calculated for each replicate determination of DOR mRNA levels. Results A Northern blot analysis using the DOR riboprobe and whole brain DOR Poly A+ RNA showed a single prominent ribonuclease resistant hybridization band of 10 kb for the mouse extract (Fig. 1, lane b). Following a longer exposure an additional minor hybridization band of approximately 12 kb in size was also observed. This minor band was not further characterized. The 10 kb long transcript was clearly the predominated hybridization band and it was used to estimate the amount of DOR mRNA. The rat extract shows two ribonuclease resistant hybridization bands, a well defined band at 12 kb and a diffuse band at 4.5 kb (Fig 1, lane c). To further confirm the specificity of the DOR riboprobe used in the solution hybridization assay, an aliquot of mouse and rat Poly A+ RNA extracts corresponding to 30 pgs of DOR sense transcript (as measured by solution hybridization) were subjected to a ribonuclease protection assay (Fig. 2). The protection assay exposes the samples to the same procedure as is used in the solution hybridization assay until the final step which involves a denaturing gel electrophoresis rather than the TCA precipitation and liquid scintillation counting step (see above). Since the DOR cDNA was cloned from the mouserat hybrid NG108-15 cell line, but originates from the mouse sequences, (10,ll) a single band the size of the riboprobe (approximately 1 kb) is protected by mouse poly A+ RNA (lane h). Neither E. coli tRNA nor rat liver RNA are able to protect the riboprobe from ribonuclease (lanes e and g). Thirty pgs of the sense standard produced a single prominent band of the expected size (approximately 1 kb) (lane f). In contrast, the heterologous Poly A+ RNA from rat provided incomplete protection of the mouse riboprobe resulting in multiple bands significantly smaller than the 1 kb protected fragment obtained with mouse Poly A+ RNA (compare lane i with lane h). A representative standard calibration curve for the DOR sense standard and mRNA is shown in Fig. 3. The curve is linear from 2 to 125 pg of the 1.08 kb sense transcript. The interassay coefficient of variation averaged 9.7% for 4 consecutive assays and the intraassay coefficient of variation for 30 duplicate sample determinations averaged 2.9% (20). From the information obtained with Northern blot analysis (Fig. 1) and the ribonuclease protection assay (Fig. 2) we were able to estimate the quantitative relationship between the signal obtained with the sense standard and the corresponding amount of the DOR mRNA. A factor (9.4) that allows conversion of the amount of sense standard to DOR mRNA in mouse CNS is obtained by multiplying the ratio of the size of the DOR message in mouse (10 kb) to the sense standard (1.08 kb) by the ratio of the size of the ribonuclease protected sense standard (1.08 kb) to the protected DOR message (1.06 kb)
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(see also 23). The lowest DOR sense calibration standard is 1.95 pg (approximately 18.3 pgs of DOR mRNA). Therefore, the lower limit of sensitivity for a sample containing 120 ugs of total RNA is 0.15 pg/ug RNA. In a concurrent solution hybridization assay the signal from a mouse or rat liver RNA extract, a tissue that does not express the DOR gene is less than 0.15 pg/ug and therefore below the limit of sensitivity of the assay.
Fig. 1 A Northern blot analysis of mouse and rat brain RNA extracts. Fifty pgs of DOR sense standard (Lane a), Poly A+ RNA from mouse (lane b, 10 ug) or rat (lane c, 15 ug) brain was separated on a 1.2% agarose gel, transferred to a nitrocellulose filter hybridized with the DOR riboprobe and then subjected to ribonuclease treatment. A single ribonuclease resistant hybridization band of 10 kb in length was detected in the mouse extract (lane b) while the rat extract contained a prominent 12 kb band and a diffuse 4.5 kb band (lane c). The film was exposed at -70” for 20 hrs (lanes a and b) and 36 hrs to visualize lane c.
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Fig. 2 Ribonuclease protection analysis of mouse and rat DOR mRNA. Following solution hybridization and ribonuclease (RNase) digestion the samples were subjected to a 4% denaturing polyacrylamide gel electrophoresis. Lanes a and b are the size markers. Lane d is the DOR riboprobe not subjected to solution hybridization. Lane c and lanes e through i contain samples that were carried through the solution hybridization and recovery steps. The sample in lane c is the riboprobe with the RNase treatment omitted, while lane e shows that E. coli t-RNA doses not protect the riboprobe from RNase treatment. Lane f shows the protection resulting from the DOR sense transcript (30 pg), lane g is rat liver RNA (100 ug), which failed to protect the riboprobe, lane h is mouse Poly A+ RNA (6 ug, equivalent to 30 pg of the DOR sense transcript), lane i is rat Poly A+ RNA (15 ug, equivalent to 30 pg of the DOR sense transcript). The arrow (lane i) indicates the largest fragment (approx. 0.56 kb) protected in rat Poly A+ RNA by the mouse derived DOR riboprobe. Lane i is from a separate but equivalent protection assay.
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I”
0
50
25 Delta
L 0
75
Transcript I
125
150
(pg) I
470
predicted
100
2349
940
f
1410
DOR mRNA (pg) Fig. 3
A standard calibration curve obtained by hybridization of the DOR riboprobe to sense standards. The calibration standards, which contained 5 ug of E. coli tRNA carrier and a known amount of DOR sense transcript, were hybridized to the DOR riboprobe and assayed using the solution hybridizationRNase protection-TCA precipitation protocol described in Materials and Methods. The ordinate shows the hybridization signal, i.e., the TCA-precipitable dpm minus background dpm (less than 1% of the total TCA-precipitable dpm of the incubated riboprobe). The abscissa indicates the pgs of DOR sense transcript present in a given calibration standard, while the lower axis shows the amounts of 10 kb DOR mRNA expected to yield the same hybridization signal as a particular calibration standard. The points are the average of two determinations and the solid line is the result of linear regression analysis. Fig. 4 shows the steady-state DOR mRNA levels in selected regions of adult mouse CNS in pg/ug RNA (SEM). The highest levels of DOR mRNA were found in caudateputarnen; CPU at 3.3 (0.1) and frontal cortex; FCtx at 2.6 (O.l), nucleus accumbens; Acb at 2.4 (0.1) and olfactory tubercle; OTu at 2.1 (0.1). DOR mRNA levels in pg/ug RNA (SEM) were for spinal cord; SpC at 1.8 (0.2), periaqueductal gray; PAG at 1.5 (0.1) and hippocampus; Hip at 1.6 (0.2). The lowest levels of DOR mRNA were measured in medial tbalamus; MThal at 0.6 (0.1) and cerebellum; Cbl at 0.6 (0.1). Mouse brain (without cerebellum) contained 2.2 (0.3) pg/ug RNA of DOR mRNA.
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Fig. 5 shows the corresponding distribution of DOR mRNA in the rat CNS in pg equivalents/ug RNA (SEM). The highest levels of DOR mRNA were measured in caudateputamen; CPU at 2.3 (0.1) while cerebral cortex; Ctx at 1.3 (O.l), brain stem; BS at 1.1 (0.1) and olfactory bulb; OB at 0.9 (0.1) contained lower levels. Hippocampus; Hip at 0.4 (O.l), cerebellum; Cbl at 0.4 (O.l), substantia nigra; SN at 0.3 (0.2) and thalamus; Thal at 0.5 (0.1) showed the lowest levels of the message. Rat brain (without cerebellum) contained 1.5 (. 18) DOR mRNA pg equivalents/ug RNA.
3.5 r
MOUSE
3.0 1 2.0 1.5 1.0 0.5 0.0 _1 Brain FCtx OTu Acb CPU Hip MThal PAG Cbl SpC
Fig. 4 The distribution of DOR mRNA in the mouse CNS. The solution hybridization assay was used to quantitate DOR mRNA distribution in whole brain without cerebellum (Brain), frontal cortex (FCtx), olfactory tubercle (OTu), nucleus accumbens (Acb), caudate-putamen (CPU), hippocampus (Hip), medial thalamus (MThal), periaqueductal gray (PAG), cerebellum (Cbl) and spinal cord (lumbar-sacral segments, SpC). Each value is the mean +S.E.M. for two separate experiments.
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2.5 -
0.0
I-
RAT
Brain Ctx OB
CPU
Hip Thai
SN
Cbl
1. BS
Fig. 5 The distribution of DOR mRNA in the rat CNS. The solution hybridization assay was used to qua&ate DOR mRNA distribution in whole brain without cerebellum (Brain), cerebral cortex (Ctx), olfactory bulb (OB), caudate-putamen (CPU), hippocampus (Hip), thalamus (Thal), substantia nigra (SN), brain stem (BS) and cerebellum (Cbl). Each value is the mean +S.E.M. for two separate experiments. Discussion Based on binding studies DOR are the most abundant of the three opioid receptor types (8). Expressed as a relative proportion, they represent 50% or more of opioid receptors in mouse and rat brain tissues (8). We have used a facile solution hybridization assay to quantitate the distribution of the DOR mR_NA in both mouse and rat CNS. Our assay is more sensitive than Northern blot analysis since up to 120 ug of target RNA can be used for each determination (20,23). Furthermore, by constructing a calibration curve from sense standards the assay is more quantitative than the usual ribonuclease protection assay. The 18s rRNA assay which measures total cellular RNA allows each sample to be normalized for the recovery of RNA and for the value of DOR mRNA to be expressed in pg (or pg equivalents) per ug of RNA. In Northern blot analysis (Fig. 1) our riboprobe hybridized to a single 10 kb transcript of DOR mRNA, which allowed the signal obtained with solution hybridization to be converted from the amount of sense standard to the corresponding amount of a 10 kb mRNA (Fig. 3). Interestingly, the single hybridization band found in mouse brain poly A+ RNA corresponds to the largest of the six DOR transcripts found in NG108-15 cells (25).
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The DOR cDNA was isolated from this mouse-rat hybrid cell line (10,ll) but appears to originate from the mouse sequences (11). In NG108-15 cell extracts this transcript represents about 36% of the total band intensity signal (20). It is not known to what degree each of the six DOR transcripts from NGlOS-15 cells are translated to yield receptor protein. However, these results suggest that the largest of the DOR transcripts, common to both NG108-15 cells and mouse brain may be of particular interest for further study. In the Northern blot analysis of rat brain poly A+ RNA extracts, two hybridization bands remained after ribonuclease treatment, a prominent 12 kb and a more diffuse 4.5 kb band. The presence of multiple transcripts in rat brain Poly A+ RNA extracts is consistent with three previous reports (3,10,15) but not with that of Abood et al. (12) who found only a single 6 kb band in rat brain RNA extracts. To confirm the specificity of our DOR riboprobe we used a ribonuclease protection assay (Fig. 2). As predicted from the relative lengths and sequences of the mouse DOR mRNA, the sense transcript and the riboprobe, the ribonuclease resistant duplex formed between the riboprobe and either the mouse DOR mRNA or the sense transcript resulted in a major band of approximately 1 kb. A single major band corresponding to a resistant duplex of the predicted length indicates that the signal obtained by solution hybridization is due to the mouse DOR mRNA present in the RNA extract and not to other RNAs that may have considerable homology to the cloned delta receptor (e.g., mu or kappa opioid receptor mRNAs) (10-16). Furthermore, using the sequence analysis software package (Genetics Computer Group, Inc., Madison, WI), we have determined that the longest stretch of perfect homology between the mouse DOR riboprobe and the mouse kappa receptor cDNA (16), rat kappa receptor cDNA (14) or rat mu receptor cDNA (13,lS) are respectively, 23, 23 and 32 bases in length. Therefore, it is very unlikely that sequences from either the mouse kappa, rat kappa or mu receptor mRNAs would contribute to the RNase protected-TCA precipitated counts. It is important to note that our RNase assay conditions result in complete hydrolysis of unprotected riboprobe and that “other” RNAs do not yield the same major protected band (Fig. 2 lanes e, g and i). In addition to a protected band of the expected size (Fig. 2 lanes f and h) an array of bands that are smaller in size were also noted. These bands result in part from thermal degradation of the riboprobe (compare Fig. 2 lanes c and d) and from the dynamics of the hybridization of RNAs. The hybrids formed between an mRNA or the sense transcript and the riboprobe do not remain as a duplex all of the time, rather they separate or “breathe” allowing RNase to attack even complementary strands, yielding some of the fragment bands shown in Fig. 2. This occurs in all protection assays where RNase is in excess (20, 23, 29). However, our solution hybridization assay compares the dpm obtained from all of the protected fragments that are trapped on the filter when the mouse DOR sense transcript is exposed to RNase with the array produced from the mouse DOR mRNA. Since both mouse DOR sense transcript and mouse DOR mRNA are exposed to the same conditions, then, as shown in Fig. 2, they yield the same array of bands. As expected, rat Poly A+ RNA extracts only partially protected the mouse DOR riboprobe as reflected in the multiple bands of 0.5 kb or less detected in a ribonuclease protection assay (Fig. 2 lane i). However, this degree of protection allowed the use of the mouse DOR riboprobe to estimate the relative amounts of the rat DOR mRNA levels (Fig. 5 and see below). Thus, solution hybridization allows a comparison of the relative abundance of DOR transcripts between tissues and/or species. For example, the NGlOS-15 cell line, which is the source of the original DOR cDNA clones (10,l l), contains six DOR transcripts that yield a mean value of 0.27 pg of DOR sense transcriptiug RNA (20). This value is very
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Delta Receptor mRNA in Mouse and Rat CNS
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similar to the mean values of 0.23 pg of DOR sense transcript/ug RNA (equivalent to 2.2 pg/ug RNA of a 10 kb DOR mRNA) found in mouse brain RNA extracts and 0.16 pg of DOR sense transcript/ug RNA (equivalent to 1.5 pg of DOR mRNA/ug RNA) found in rat brain RNA extracts. Furthermore, the solution hybridization assay can be used to complement and extend mRNA distribution maps obtained by in situ hybridization (30). In agreement with Mansour et al., (9) we found the highest levels of DOR mRNA in mouse telencephalic structures of the limbic system including caudate-putamen, frontal cortex, olfactory tubucle, nucleus accumbens and hippocampus (Fig. 4). An excellent correlation is also obtained when the distribution of DOR binding sites in the limbic system, as measured by autoradiography (7-9), is compared with the distribution DOR mRNA measured by in situ hybridization (9) or solution hybridization (this report). This colocalization of DOR mRNA and receptors in the limbic system may permit regulation of the endogenous opioid system by activation of autoreceptors and/or via local opioidergic circuits and may be reflected in the ability of DOR selective opioids to elicit seizures (26,27) and to facilitate reward behavior (28). DOR mRNA, as expected from the results of Mansour et al., (9), was measured in the mouse spinal cord, hippocampus and whole brain (Fig. 4). However, in contrast to report of Mansour et al., (9) we were able to measure DOR mRNA in periaqueductal gray (PAG) and cerebellum. While these differences may result from the different but partially overlapping probes used in our study compared to that of Mansour et al., (9), it is more likely that our ability to increase the amount of target RNA in the solution hybridization assay accounts for the greater sensitivity (see below). In this regard it must be emphasized that our assays include a comparable amount of an RNA extract from a tissue, e.g., (mouse or rat liver) that does not express the DOR and a complete standard calibration curve so that the lower limit of sensitivity can be accurately defined for each assay. Autoradiographic studies (7) have measured DOR binding in the mouse PAG so that our results (Fig. 4) support colocalization of DOR mRNA and receptor in this mesencephalic area involved in the processing of nociceptive information (7). We attempted to qua&ate DOR mRNA levels in selected rat CNS regions with the mouse DOR riboprobe. When there is a high degree of homology in the mRNA sequences from the two species, the solution hybridization procedure can be used to measure relative mRNA levels using a heterologous probe (22,29). In ribonuclease protection assays the mouse riboprobe protected multiple bands from rat brain extracts indicating the expected sequence variation between the two species (Fig. 2). The mouse riboprobe is not completely protected by rat DOR mRNA so that our assay yields values for the rat DOR mRNA which are underestimates of the true amount. Since it is not possible to determine the exact pg amounts, the rat levels are expressed as pg equivalents which allows a relative comparison of the signal obtained from different rat CNS areas (22,29). With Northern blot analysis the mouse riboprobe hybridized to transcripts from rat brain extracts that are 12 and 4.5 kb in length and clearly a different size than the mouse DOR mRNA (Fig. 2). Since the Northern blot analysis utilized a ribonuclease treatment, the signal from rat brain which remained, represents hybridization of the rat DOR sequences with the mouse DOR riboprobe. As with the mouse, we selected for analysis rat CNS regions that have been shown by autoradiography (1,2) and in situ hybridization (30) to contain a relatively high (e.g.,
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caudate-putamen and olfactory bulb) or low (e.g., thalamus and cerebellum) density of DOR and/or DOR mRNA. As with the distribution of the mouse DOR mRNA we found that rat DOR mRNA levels are 3 to 8 fold higher in telencephalic regions than in the other brain regions we sampled (Fig. 5). These results, although limited, suggest that like the mouse DOR mRNA, a close correspondence exists between the density of DOR receptors and DOR mRNA expression in the rat CNS. Acknowledgements Supported in part by NIDA Grant DA01457 (CEI), NIDA Training Grant DA07274 (SJ). CEI is a recipient of a Research Scientist Award from NIDA (DAO0198). BK is an Aaron Diamond Foundation Fellow and this work was supported in part by The Aaron Diamond Foundation.
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