Chang, K.-J., Hazum, E. & Cuatrecasas, P. (1979) Mol. Pharmacol. 16, 91-104. 10. Goodman, R. R., Snyder, S. H., Kuhar, M. J. & Young,. W. S. (1980) Proc. Natl.
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6667-6671, October 1985 Medical Sciences
Visualization of IAl opiate receptors in rat brain by using a computerized autoradiographic subtraction technique (morphine/enkephalin)
ROBERT R. GOODMAN AND GAVRIL W. PASTERNAK The Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, and Departments of Neurology and Pharmacology, Cornell University Medical College, New York, NY 10021
Communicated by Max Tishler, May 13, 1985
ABSTRACT We have developed a quantitative computerized subtraction technique to demonstrate in rat brain the regional distribution of #1 sites, a common very-high-affinity binding site for both morphine and the enkephalins. Low concentrations of [D-Ala2, D-Leu5]enkephalin selectively inhibit the ,., binding of [3Hjdihydromorphine, leaving Is2 sites, while low morphine concentrations eliminate the ,u binding of [3H][D-Ala2, D-Leu5]enkephalin, leaving 8 sites. Thus, quantitative differences between images of sections incubated in the presence and absence of these low concentrations of unlabeled opioid represent Au binding sites. The regional distributions of ,i1 sites labeled with [3H]dihydromorphine were quite similar to those determined by using [3H][D-Ala2, D-Leu5Jenkephalin. High levels of ,u, binding were observed in the periaqueductal gray, medial thalamus, and median raphe, consistent with the previously described role of ,sl sites in analgesia. Other regions with high levels of ,u binding include the nucleus accumbens, the clusters and subcallosal streak of the striatum, hypothalamus, medial habenula, and the medial septum/diagonal band region. The proportion of total specific binding corresponding to I,1 sites varied among the regions, ranging from 14% to 75% for [3H][D-Ala2, D-Leus]enkephalin and 20% to 52% for [3H]dihydromorphine.
levels of /u1 binding (36, 37). However, this technique is tedious and not applicable for widespread mapping of 1,u sites. Therefore, we developed a computerized pixel-by-pixel subtraction technique for digitized images that enables the computer to generate an image of the inhibited binding. Using this technique, we now report the distribution of ul binding sites, using both a ,u and a 8 3H-labeled ligand at three levels of the rat brain. A preliminary report ofthese results has been presented (38). METHODS Tissue preparation and incubations were performed by using modifications of the technique described by Young and Kuhar (6). Coronal sections (8 ,um thick) from male SpragueDawley rats (180 g) were incubated for 15 min in Tris HCl buffer (0.17 mM; pH 7.7 at 25°C) containing 50 ,uM GTP and 100 mM NaCl to remove endogenous opioids and then washed in Tris buffer alone for 5 min. Groups of adjacent sections were then incubated for 90 min at 25°C in Tris buffer with [3H]dihydromorphine (1 nM; 80.3 Ci/mmol; New England Nuclear; 1 Ci = 37 GBq) in the absence and presence of unlabeled [D-Ala2, D-Leu5]enkephalin (5 nM); or with [3H][D-Ala2, D-Leu5]enkephalin (1 nM; 43.6 Ci/mmol; New England Nuclear) in the absence or presence of morphine sulfate (5 nM). Nonspecific binding was determined from sections incubated with the radioligand and levallorphan (1 ,uM). A section from each group was stained with hematoxylin and eosin for use in identifying anatomical regions. Slides were washed twice for 5 min each in Tris buffer (0°C), dipped once in distilled water, dried with a stream of cold dry air, placed in an x-ray film cassette along with sections containing known concentrations of tritium (dpm/mg of tissue, dry weight), exposed to LKB Ultrofilm at 4°C with desiccant for 6 months, and developed. Standards were prepared as previously described (39). The autoradiographs of the tissue sections and standards were digitized by using a Vidion-based EYECOM II image processing system linked to a PDP11/23 computer. Multiple standards on each film varied by less than 5%. The correlation of optical densities of the standards to their activity levels (dpm/mg of tissue) were fit to a polynomial equation and used to calculate the concentrations of radioligand in the tissue sections (fmol/mg of tissue). Selected regions of the experimental sections were identified on adjacent sections stained with hematoxylin and eosin. Results represent averages of values determined in two to four areas of two sections of a rat brain. Similar relative values were obtained in a second rat brain. For digital subtractions, images were aligned and all optical densities were converted to activities (dpm/mg of tissue). Pixel-by-pixel subtractions of the activities were then performed and the differences were converted back into optical densities and displayed as a separate image. All images represent only specific binding. Images of i,, and /2, li, and
Autoradiography provides an effective means to accurately map opiate-binding sites within the rat brain and correlate their distribution with brain regions known to mediate opiate actions (1-6). In addition, autoradiographic studies have greatly strengthened the suggestion that there are distinct ,u and 8 receptors (7-9), by demonstrating their unique regional distributions within the brain (10, 11). Recently, we proposed that morphine and the enkephalins label three, not two, subpopulations of sites (12, 13). In addition to the enkephalinpreferring 8 sites and the classical morphine-selective (p.2) sites demonstrated in the guinea pig ileum (7, 14), there is a common site (p.l) to which both morphine and the enkephalins bind with very high affinity. This p.l site, corresponding to the high-affinity site identified in 1975 (15), differs from the pU2 and 8 sites developmentally, phylogenetically, biochemically, and pharmacologically, playing a major role in a number of important opiate actions, including supraspinal analgesia, but not in others, such as respiratory depression and most signs of physical dependence (12-14, 16-34). Homogenate binding studies reveal a heterogeneous distribution of Al sites in rat brain (31, 35), but they lack the necessary detailed resolution. Autoradiography, on the other hand, faced a number of difficulties, including the lack of a ligand that could selectively label pul sites. Competition approaches can selectively inhibit p,1 binding, but visualizing the lost binding is difficult. Moskowitz and Goodman utilized grain counting in several regions of mouse brain to determine The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Medical Sciences: Goodman and Pasternak
8, I2 alone, and 8 alone were obtained by subtracting images from sections incubated with levallorphan. ttl images were determined by subtracting the images obtained in the presence of 5 nM competitor from total binding. Although quenching can vary among regions (40), we did not defat the sections, since this technique can decrease specific binding by over 50% and we did not wish to risk a differential loss of the binding subtypes.
Proc. Natl. Acad. Sci. USA 82
(1985)
Morphine Enkephalin
aL receptor
RESULTS Homogenate binding studies have suggested that morphine and the enkephalins bind to three sites: (i) the morphineselective A2 site, (it) the enkephalin-selective 8 site, and (iii) a common, very-high-affinity, Al site (Fig. 1). To establish the regional distribution of each of these sites, we used several binding conditions. First, we measured the total binding of each radioligand as well as nonspecific binding, defined as the labeling remaining in the presence of levallorphan (1 MkM). The autoradiogram of binding in the presence of levallorphan was then subtracted from the autoradiogram of total binding to obtain the distribution of total specific [3H]dihydromorphine binding, consisting of both pu and &2 sites. The distribution of total specific [3H]dihydromorphine binding (Fig. 2) was very similar to that previously reported (10). Relatively high levels of binding were observed in lamina IV of the cortex, the clusters and subcallosal streak of the striatum, the nucleus accumbens, the medial thalamus, hypothalamus, amygdala, periaqueductal gray, medial habenula, superior colliculus, and the median raphe. Quantification of binding in these regions is presented in Table 1. To determine the separate distributions of Al and A2 sites, we incubated an adjacent section with [3H]dihydromorphine and unlabeled [D-Ala2, D-Leu5]enkephalin at a concentration (5 nM) sufficient to inhibit the Al binding of [3H]dihydromorphine without significantly affecting the A2 sites (12). The distribution of 12 sites was then determined by subtracting the nonspecific binding from this autoradiogram. Overall, the regional distribution of A2 sites was quite similar to total specific [3H]dihydromorphine binding. Having established the binding of /il and 92 sites together and A2 sites alone, we next examined the localization of Al sites. Pixel-by-pixel computerized subtraction of the autoradiogram of [3H]dihydromorphine binding in the presence of unlabeled [D-Ala2, D-Leu5]enkephalin (I.2) from total specific [3H]dihydromorphine binding (Al and 2) produced an image of 1 sites. The images of 1, binding revealed low levels in the cortex and high values in the medial septal nucleus/nucleus of the diagonal band, nucleus accumbens, clusters of the caudate, medial thalamus, hypothalamus, medial habenula, superior colliculus, periaqueductal gray, and median raphe (Table 1). Although the overall distributions of tul and A2 sites appeared similar, Atl binding as the percentage of total [3H]dihydromorphine binding varied from region to region. The enkephalin lowered binding in lamina IV by 24% and in the pyramidal layer of the hippocampus by 27% compared to more than 50% of total specific binding in the medial thalamus and the ventral periaqueductal gray. Subtracting images of nonspecific binding from total [3H][D-Ala2, D-Leu5]enkephalin binding yielded total specific [3H][D-Ala2, D-Leu5]enkephalin binding (tti + 8). Adjacent
t2 receptor
5receptor FIG. 1. Schematic representation of the binding of morphine and enkephalins to a,, A2, and 8 binding sites.
sections were incubated with [3H][D-Ala2, D-Leu5]enkephalin in the presence of morphine at a concentration sufficient to eliminate the g, binding, leaving only 8 binding. Our autoradiograms of [3H][D-Ala2, D-Leu5]enkephalin binding in the presence of morphine were very similar to those in earlier studies that also included a low dose of morphine (10). High levels of binding were noted in the cortex, olfactory tract, nucleus accumbens, claustrum, and lateral striatum (Fig. 2). In the absence of morphine, the distribution of [3H][D-Ala2, D-Leu5]enkephalin binding was quite different. These conditions, reflecting the labeling of both 1,u and 8 sites, clearly demonstrated significant binding in the nucleus accumbens, the medial thalamus, the superior colliculus, periaqueductal gray, the clusters and subcallosal streak of the corpus striatum, medial septum/diagonal band area, medial habenula, amygdala, and median raphe. The differences in labeling between the sections incubated with [3H][D-Ala2, D-Leu5]enkephalin alone and in the presence of morphine were primarily due to a, binding. The computer-generated images of the pixel-by-pixel subtractions were quite similar to those of ul binding generated by using [3H]dihydromorphine. High levels of binding were found in the nucleus accumbens, clusters of the striatum, subcallosal streak, medial thalamus, periaqueductal gray, superior colliculus, medial habenula, hypothalamus, amygdala, and median raphe. The percentage of total specific [3H][D-Ala2, D-Leu5]enkephalin binding corresponding to Al sites varied markedly among the regions. g, binding represented about 15% of total binding in the pons and 20% in the olfactory tract and lateral striatum. In contrast, Al sites made up 75% of total binding in the medial septal nuclei/nucleus of the diagonal band, 70% in some areas of the thalamus, and 60% in the hypothalamus, superior colliculus, and periaqueductal gray (Table 1). If gu sites truly represent a common site for both the enkephalins and morphine, labeling with either [3H]dihydromorphine or [3H][D-Ala2, D-Leu5jenkephalin should produce similar distributions of gu sites. Qualitatively, the images of ,41 sites labeled with the two radiolabeled compounds were quite similar. We extended this comparison of tL1 binding by
FIG. 2. Computerized autoradiograms of gl, pa, and 8 binding sites. Adjacent tissue sections taken through the striatum (A), the thalamus (B), or the brainstem (C) were prepared and incubated as described in Methods. In all three levels, the first image in the top row is the total specific binding of [3H]dihydromorphine, representing both ,uI and /.2 binding. The second image is the specific binding remaining in the presence of unlabeled [D-Ala2, D-Leu5]enkephalin (5 nM), corresponding to /A2 sites. The third image is the computer-gene'rated subtraction of the first two and corresponds to ;Al binding. The first image in the second row is total specific [3H][D-Ala2, D-Leu5]enkephalin binding (g, + 8) and the second is the specific binding remaining in the presence of morphine (5 nM), corresponding to 8 sites. The third image is the computer-generated distribution of Al sites obtained from the difference between the first and second image.
Proc. Natl. Acad. Sci. USA 82 (1985)
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Medical Sciences: Goodman and Pasternak
Proc. Natl. Acad Sci. USA 82
Table 1. Quantitative distribution of 3H-labeled opioid binding to /Al, g2, and 8 sites [3H]Dihydromorphine Binding, fmol/mg tissue binding, a, Region /2 % of total n Al + A2 Al Temporal cortex Lamina I 1.9 ± 0.3 1.4 ± 0.1 0.5 ± 0.2 24 ± 8 3 Laminae II/III 1.5 ± 0.1 1.4 ± 0.1 0.1 ± 0.1 7± 8 3 Lamina IV 3.4 ± 0.4 2.6 ± 0.4 0.8 ± 0.1 24 ± 4 3 LaminaV 2.5 ± 0.1 2.0 ± 0.2 0.5 ± 0.1 20 ± 5 3 LaminaVI 2.3 ± 0.2 1.8 ± 0.2 0.5 ± 0.1 23 ± 4 3 Lateral olfactory tract 3.7 ± 0.6 2.2 ± 0.4 1.5 ± 0.4 40 ± 9 4 Medial septum/nucleus of the diagonal band 12.3 ± 2.3 7.0 ± 1.4 5.3 ± 1.2 43 ± 5 4 Nucleus accumbens 15.4 ± 2.8 7.6 ± 0.8 7.8 ± 2.2 4 50 ± 6 Hippocampus 2.9 ± 0.2 2.1 ± 0.3 0.8 ± 0.2 27 ± 8 4 Claustrum 7.4 ± 0.7 4.4 ± 0.1 3.0 ± 0.8 Amygdala 40 ± 7 3
Striatum/caudate Clusters Intercluster Medial Lateral Thalamus Centromedian Rhomboid Ventrolateral Hypothalamus Ventromedial Lateral Medial habenula Superior colliculus Periaqueductal gray Dorsal Ventral Raphe Dorsal
(1985)
D-LeuJ]Enkephalin Binding, fmol/mg tissue binding, [3 H][D-Ala2,
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0.7 1.1 1.0 0.8 0.7 0.2
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49 ± 43 ± 45 ± 52 ± 35 ± 21 ±
20 4 7 5 5 3
4 4 4 4 4 2
6.1 ± 9.3 ± 3.4 ± 8.5 ± 6.7 ±
0.2 1.5 ± 0.6 4.6 ± 0.4 0.7 4.5 ± 0.2 4.8 ± 0.6 0.3 1.5 ± 0.1 1.9 ± 0.2 0.6 6.0 0.2 2.5 ± 0.6 1.2 3.3 ±0.7 3.4 ± 0.6
75 ± 52 ± 48 ± 29 ± 51 ±
8 3 6 5 3
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4
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6.2 ± 0.4
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4
1.0 ± 0.3 0.7 ± 0.3
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4 4
6.7 ± 0.3 4.7 0.2 10.6 ± 1.2 8.0 ± 0.4
2.0 ± 0.3 2.6 ± 1.0
31 ± 4 24 ± 6
4 4
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52 ± 1 51 ± 4
2 2
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2.8 ± 0.6 2.7 ± 0.4 1.5 ± 0.2
6.7 ± 0.7 5.4 ± 0.8 3.4 ± 0.2
71 ± 3 67 ± 1 69 ± 3
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1.9 1.8 3.2 1.5
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1.9 ± 0.2 1.4 ± 0.3
3.7 4.1 6.6 3.0
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0.3 2.6 ± 0.2 0.4 3.1 ± 0.6 1.5 5.0 ± 0.8 0.4 2.5 ± 0.5
3.0 ± 0.8 2.5 ± 0.8
2.6 ± 0.3 2.6 ± 0.4
46 ± 7 52 ± 12
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1.5 ± 0.4 3.6 ± 0.8
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0.5 0.7 1.2 0.4
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3.3 ± 0.1 1.4 ± 0.2 1.9 ± 0.2 57 ± 8.1 ± 0.8 3.6 ± 0.4 4.5 ± 0.4 56 ± 2.4 ± 0.5 2.1 ± 0.4 0.3 ± 0.1 14 ± Pons Images were quantified and the appropriate subtractions were performed as described in Methods. Values represent the mean ± specific binding for n determinations.
Median
n
correlating quantitative levels of A binding determined with the two radioligands in the various regions (Fig. 3). The levels of tLj labeling correlated quite well (r2 = 0.8), with a slope (1.2) near unity.
DISCUSSION Binding experiments have identified a very-high-affinity site labeled by both morphine and the enkephalins more potently than the sites selective for either compound alone (12, 16, 17, 19, 32, 33). Pharmacological studies have implicated tL1 sites in a number of opiate actions, including supraspinal analgesia (12-14, 16-34). We therefore wanted to determine if Lu sites were localized to brain regions known to be important in opiate analgesia, such as the periaqueductal gray. In addition, we wanted to determine if both tL and 8 3H-labeled ligands yielded the same distribution of tL1 sites within the brain, as predicted by our hypothesis of a common site for both classes of compounds. The lack of a radioligand able to selectively label ul sites coupled with their low density within the brain presented problems. Competition studies can selectively inhibit the tL1 binding of either 1L or 8 radioligands relatively selectively, but to generate an image of these sites, we had to develop a computerized pixel-by-pixel subtraction technique. The hypothesis of a common site for both morphine and the enkephalins predicts that radioligands from either the g or the
6 1 4
2 2 4 SEM of
8 class should be able to label ,l sites equally well. We therefore examined ul binding by using the 1L drug [3H]dihydromorphine and the 8 ligand [3H][D-Ala2, D-Leu5]enkephalin. Simply obtaining a subtracted image with only one ligand was, in our opinion, not sufficient. The computergenerated Al images from the two 3H-labeled ligands were remarkably similar at all three levels. A number of regions possessed high levels of Al binding, but the most intriguing areas included the medial thalamus, the periaqueductal gray, and the median raphe, all of which are known to play a major role in the modulation of pain. Other areas also contained high levels of pul binding, but their significance is less clear. The distribution of ul sites observed in our studies was similar to that deduced from homogenate binding studies examining the sensitivity of 3H-labeled opioid binding to naloxazone (31) and examining direct labeling by [3H]naloxonazine (35). Autoradiographic studies of selected regions of mouse brain by using a grain-counting technique have also attempted to quantitate ul binding and have yielded values very similar to ours (36, 37). Of particular interest was the comparison of pu sites in the periaqueductal gray of C57BL/6BY and CXBK mice. The levels of g, sites in the CXBK mice, a strain of mice relatively insensitive to morphine analgesia, were less than half levels found in the morphine-sensitive C57BL/6BY strain. These findings support the association between ul sites and supraspinal analgesia (18, 20-25, 30). Very low levels of ,ul sites were
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FIG. 3. Correlation of lu binding of either [3H]dihydromorphine or [3H][D-Ala2, D-Leu5]enkephalin in various brain regions. Levels of iu binding determined with [3H]dihydromorphine were compared to values determined with [3H][D-Ala2, D-Leu5]enkephalin ([3H]DADLE). Linear regression analysis reveals a correlation coefficient (r) of 0.9 and a slope of 1.2.
detected in the dorsal nucleus of the vagus or the nucleus of the solitary tract, corresponding well to our recent studies reporting little involvement of Al sites in morphine-induced respiratory depression (21, 22). The quantitative levels of lu binding in regions obtained with the two 3H-labeled ligands also correlated quite well. The differences might result from slight regional differences between the two sets of adjacent sections, slight differences in affinity of the two 3H-labeled ligands yielding differing percentages of receptor occupancy, and possibly inhibition of small amounts of 8 and 2 sites, respectively, by morphine and [D-Ala2, D-Leu5]enkephalin at 5 nM. The percentage of total specific binding corresponding to Ali sites varies from region to region for both 3H-labeled dihydromorphine and the enkephalin. Although the differences were most dramatic for the [3H]enkephalin, ranging from 14% to 75%, [3H]dihydromorphine binding also had prominent differences, from approximately 25% to over 50%. Moskowitz and Goodman also noted that aul sites represented variable proportions of total specific binding in various brain regions. ul accounted for high percentages of total [3H]dihydromorphine binding in the nucleus of the diagonal band (36%), striatal patches (64%), the nucleus accumbens (67%), the central nucleus of the amygdala (49%), the ventral periaqueductal gray (55%), and laminae I/II ofthe spinal cord (62%). These values are very similar to ours. Although the general distributions of ul and J2 sites appear quite similar in the computer-generated images, these regional differences support the concept of a distinct receptor subtype. We thank Drs. J. B. Posner and W. R. Shapiro for their support of these studies. We also gratefully acknowledge the invaluable assistance of Mr. Peter Sher, who developed the software used for the subtraction technique. These studies were supported, in part, by grants from the National Institute on Drug Abuse (DA02615) and the American Cancer Society (PDT169) to G.W.P. as well as a core grant from the National Cancer Institute (CA08748) to Memorial Sloan-Kettering Cancer Center.
12.
13. 14. 15.
16. 17. 18.
19. 20. 21. 22.
23. 24. 25. 26. 27.
28. 29. 30. 31.
32. 33.
34.
6671
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400-405. 35. Johnson, N., Houghten, R. & Pasternak, G. W. (1982) Life Sci. 31, 1381-1384. 36. Moskowitz, A. S. & Goodman, R. R. (in press) Brain Res. 37. Moskowitz, A. S. & Goodman, R. R. (in press) Brain Res. 38. Goodman, R. R. & Pasternak, G. W. (1984) Soc. Neurosci. Abstr. 10, 586 (abstr.). 39. Unnerstall, J. R., Neihof, D. L., Kuhar, M. J. & Palacios, J. M. (1982) J. Neurosci. Methods 6, 59-73. 40. Herkenham, M. & Sokoloff, L. (1984) Brain Res. 321, 363-368.
