Physiology&Behavior,Vol. 51, pp. 1093-1096, 1992
0031-9384/92 $5.00+ .00 Copyright© 1992PergamonPress Ltd.
Printed in the USA.
BRIEF COMMUNICATION
Comparison of # Opioid Receptors in Brains of Rats Bred for High or Low Rate of Self-Stimulation R U T H G R O S S - I S S E R O F F , *~ E D N A COHEN~" A N D Y E H U D A S H A V I T t
*Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel and tDepartment of Psychology, The Hebrew University of Jerusalem, Jerusalem 91905, Israel Received 6 M a y 1991 GROSS-ISSEROFF, R., E. COHEN AND Y. SHAVIT. Comparisonof a opioid receptorsin brains of rats bredfor high or low rateofself-stimulation. PHYSIOL BEHAV 51(5) 1093-1096. 1992.--Opiates and endogenous opioid peptides play an important role in reward-mediated behaviors, including self-stimulation. Two strains of rats, LC2-Hi and LC2-Lo, selectivelybred for high vs. low rate of lateral hypothalamic self-stimulation, were employed in the present study. Quantitative autoradiography was performed on brains of adult male rats of each strain, using the a opioid receptor agonist 3H-DAGO. Strain differences in receptor density were observed in the nucleus accumbens and in ventral areas of the hippocampus. ~H-DAGO Autoradiography Nucleus accumbens
Self-stimulation
Genetic strains
ELECTRICAL stimulation of certain brain regions can be rewarding, as is evident from the phenomenon of self-stimulation (23). Intracranial self-stimulation (ICSS) is believed to involve the activation of central mechanisms which, under normal circumstances, subserve the processing of natural reward signals (23,25,27,38). Evidence from various studies, including studies of self-administration (10) and conditioned place preference (3,21,35), suggests that opiates and endogenous opioid peptides can also be rewarding [for a recent review see (37)]. It has been suggested that central reward mechanisms have genetic determinants (4,13,16,22). Several strains of rats, that differ in their response to lateral hypothalamic rewarding stimulation, have been selectively bred at the Hebrew University of Jerusalem (l 3,14). Two of these strains, LC2-Hi and LC2-Lo, were derived from a common ancestry and subjected to bidirectional selective pressure for high and low rates of ICSS, respectively. Consistent differences in rates of bar pressing for rewarding brain stimulation have been reported in these two strains (14). As demonstrated by further studies, other motivational differences evolved between these strains, concurrent with those related to ICSS. Compared with LC2-Lo, the LC2-Hi rats show more pronounced preference for sweet solutions (8), higher in-
~ opioid receptors
Reward
cidence of eating induced by lateral hypothalamic stimulation (34), and more robust morphine tolerance following prolonged maintenance on saccharin solution (15). In view of the possible involvement of endogenous opioids in reward associated with feeding (28,39), intake of sweet substances (5,15,17) and self-stimulation (1,7,19,29,32,36), it was hypothesized that the two strains might differ in the activity of their endogenous opioid systems. While there are obvious methodological difficulties in studying the correlation between motivational and other tendencies using a within-subject design, there is a clear advantage in using strain comparisons for this purpose [see (12,13)]. The differences previously established concerning the LC2 strains permit us to study endogenous opioid mechanisms in intact and experimentally naive rats whose ICSS characteristics are known (within the limits of a probabilistic estimation) without having been actually measured. Any difference found between these strains might be logically related to differences in their readiness to perform self-stimulation. Strain differences in the density of # opioid receptors were found in a preliminary saturation binding study, performed on homogenates of discrete brain areas taken from LC2-Hi and
Requests for reprints should be addressed to Ruth Isseroff, Biological Psychiatry Laboratory, Psychiatry Wing, The Chaim Sheba Medical Center, Tel Hashomer, Israel.
1093
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G R O S S - I S S E R O F F , (?OHEN A N D S H A V I I
TABLE 1 DISTRIBUTION OF tL OPIOID RECEPTORS IN LC2-HI AND LC2-LO RATS
Re,on
LC2-Hi [3H]DAGO bound
LC2-Lo
[~H]DAGO
LC2-t,o/
bound
LC2-Hi
309 _+ 65 [6] 381 ± 49 [6]
329 ± 31 [7] 363 ± 50 [7]
1.06 0.95
254 +_ 40 [7] 283 ± 57 [7]
236 ± 36 [7] 261 +_ 24 [7]
0.93 0.92
228 ± 31 [7] 246 _+ 34 [7]
195 +_ 28 [7] 191 _+ 38 [7]
0.86 0.78
218 219 204 197 897
[7] [7] [7] [7] [7]
0.95 1.04 0.86 0.76 0.85
476 ± 53 [7] 246-+ 29[7]
443 ± 60 [7] 2 1 7 ± 5t [7]
0.93 0.88
396 ± 51 [7] 271 ± 25 [7] 494 ± 102 [7]
382 ± 39 [7] 242 _+ 39 [7] 304 _+ 53 [7]
0.96 0,89 0.62
760 ± 70 [6] 609 ± 73 [6]
546 +_ 112 [6] 366 ± 82 [6] 518 310 352 957
:
~
t~
0.72 0.60
1.63 2.20
10 10
0.136 0.052
[6] [6] [6] [8]
0.68 0.52 0.50 0.81
2.16 3.30 2.23 1.7l
10 10 10 13
0.056 0.012 0.050 0.110
353 +_ 31 [7] 64 ± 5 [7] 645 -+ 80 [7]
455 _+ 41 [8] 84 ± 9 [7] 880 ± 43 [6]
1.29 1.31 1.36
1.89 1.97 2.45
13 12 11
0.082 0.072 0.032
592 -+ 89 [7] 188 -+ 22 [5] 208 -+ 47 [7]
390 ± 90 [8] 252 -+ 38 [8] 289 ± 104 [8]
0.65 1.34 1.38
480 _+ 124 [5] 434 ± 53 [6]
476 -+ 79 [7] 460 -+ 57 [6]
0.99 1.05
Cortex
Agranular insular Lateral orbital Frontal Area l Area 2 Somatomotor Forelimb area Hindlimb area Occipital Area l, binocular Area 1, monocular Area 2, lateral Area 2, mediolateral Presubiculum Hippocampus Dorsal CA fields Pyramidal layer Molecular layer Dentate gyrus Granular layer Molecular layer Hilus Ventral CA fields Pyramidal layer Molecular layer Dentate gyrus Granular layer Molecularlayer Hilus Amygdala Corpus striatum Caudate-putamen Globus pallidus Accumbens Thalamus Posterior nuclear group Ventrolateral nucleus Ventral posteromedial nucleus Hypothalamus Lateral Substantia Nigra
229 210 236 260 1061
_+ 34 _+ 36 ± 25 ± 21 ± 229
765 ± 594 -+ 706 ± 1180±
[6] [7] [6] [6] [6]
85 [6] 81 [6] 144 [6] 83 [7]
± ± ± +_ -+
-+ ± _+ ±
52 45 38 38 32
77 48 65 98
Table presents mean +_SEM specific [3H]DAGO binding in brains (number in brackets), expressed as fmol [3H]DAGO/mg protein, as determined by quantitative autoradiography. LC2-Lo/LC2-Hi--the proportion between the means of the two strains, t--value of t in a student's t-test; dr-degrees of freedom; p--probability of t in a two-way student's t-test. LC2-Lo rats. Bm~x values, obtained from Scatchard plots of tritiated D A G O b i n d i n g experiments, were significantly higher in the corpus striatum a n d thalamus of LC2-Lo brains, as compared with the respective regions o f LC2-Hi brains (p < 0.01, 2-tailed t-tests for m a t c h e d observations). No appreciable strain differences were observed in the hindbrain, midbrain, hypothalamus, h i p p o c a m p u s a n d anterior cerebral cortex (26). T h e present study was designed to confirm a n d extend these preliminary results. G i v e n the high resolution offered by the
quantitative autoradiography method, a n a t t e m p t was m a d e to p i n p o i n t differences in substructures within the gross regions excised for the h o m o g e n a t e experiments. The forebrain was chosen as a first target, in view of the previous findings. Eight LC2-Lo a n d 7 LC2-Hi adult male rats were used. Following decapitation brains were quickly removed, split at the midsagittal plane, frozen o n dry ice, a n d t h e n stored at - 7 0 ° C . T h e right cerebral hemisphere o f each a n i m a l was sectioned at - 1 5 ° C on a cryotome. Approximately 3 0 - 3 5 sagittal slices, 30
AUTORADIOGRAPHY OF # OPIOID RECEPTORS IN TWO RAT STRAINS #m thick, were obtained from each brain. Slices were thaw mounted on gelatin coated glass slides, kept overnight at - 2 0 ° C and then stored at - 7 0 ° C until use. Slides were incubated for 60 min at 25°C, with 1 nM 3H-DAGO (Tyr-D-Ala-Gly-Nmethyl-Phe-Gly-ol) (N.E.N., 58 Ci/mmol) in 50 mM Tris-HC1 buffer, pH 7.4 (9). Nonspecific binding was determined in the presence of 1 tiM etorphine. Slides were then washed for 9 min in the same ice cold buffer, dipped in ice cold water to remove buffer salts, dried on a 50°C hot plate and apposed to tritium sensitive film (Amersham 3H-Hyperfilm) for 12 weeks. The film was coexposed to commercial (Amersham) tritiated standards. It was manually developed and fixed with Kodak developer and fixer. Sections were stained with cresyl violet for anatomical reference; the Paxinos and Watson (24) rat brain atlas was used for identification of structures. Under the above conditions, nonspecific binding was at the level of film background. Autoradiograms were analyzed using an IBM-PC based computerized image analysis system with a PC-vision digitizing board (Imaging Technology, Inc.) and customized software. Standardization curves derived from the tritiated standards were used to convert gray level readings of the autoradiograms into equivalents of fmol 3H-DAGO/mg protein. Measurements of each region, within each brain, were done at least in duplicate. Mean values of 3H-DAGO binding, obtained from 30 brain regions and averaged within each strain, are presented in Table 1. As expected, we found a highly heterogeneous distribution of opioid receptors in the rat brain. High density of binding appeared over the amygdala, nucleus accumbens, and hypothalamus, whereas lower levels were found in the cortex, thalamus, and hippocampus. Heterogeneity was also observed within several of the brain regions studied: Receptor density was higher in the dorsal hippocampus than in the ventral hippocampus; receptors were more abundant in frontal areas of the cortex than in the occipital cortex; the caudate-putamen complex showed considerable binding, with a characteristic patchy pattern, while the globus pallidus had relatively low binding. The observed distribution was in good agreement with previously published results (20,31 ). Respective brain regions of the two strains were compared using 2-tailed t-tests; the results are presented in Table 1. In accordance with the results of the preliminary homogenate study, a higher 3H-DAGO binding was found in striatal structures of the LC2-Lo strain, as compared to the LC2-Hi respective structures. Significant strain differences in the predicted direction were observed in the nucleus accumbens. Similar differences were detected in the other striatal structures, the caudate-putamen and the globus pallidus; these differences, however, failed to reach statistical significance. Strain differences in the opposite direction were found in the ventral hippocampus. ~H-DAGO binding in the molecular layer
1095
and hilus of the ventral dentate gyrus were significantly higher in LC2-Hi than in LC2-Lo rats. A similar (but not significant) trend appeared in the granular layer of the ventral dentate gyrus and in the molecular layer of the ventral CA fields. No appreciable strain differences were detected in structures of the dorsal hippocampus. This might probably explain the absence of any indication of strain differences in assays of the entire hippocampal tissue in the previous study. Strain differences, which were previously observed in homogenates of the thalamus, were not confirmed by the present study. None of the other regions examined has shown significant strain differences in 3H-DAGO binding. The results of the autoradiography experiments do not allow to determine whether the differences in 3H-DAGO binding, observed between the two strains, are due to differences in affinity (Kd) or in U receptor density (Bmax), although the previous data of the saturation binding experiments (26) would favor the latter possibility. The most interesting finding reported here is the higher density of ~ opioid receptors in the nucleus accumbens of LC2-Lo compared with LC2-Hi rats. The nucleus accumbens is rich in opioid peptides (18) and opioid receptors (20,31); this structure has been associated with the mediation of the reinforcing effects ofopioids (33,36). Nucleus accumbens self-stimulation is attenuated by opiate antagonists (29,32,38). Based on findings of earlier studies, it has been suggested that the LC2-Lo strain is less hedonic and less responsive to incentive stimuli, compared with the LC2-Hi strain (8,13,15,34). The finding of higher density of opiate receptors in the nucleus accumbens of LC2-Lo rats might reflect lower activity of opioid peptides, or, paradoxically, an internal reward system which is more efficient and, therefore, less dependent on external (electrical, gustatory or other) stimulation. Further studies are needed to elucidate this point. The finding of higher ~ opioid receptor binding in the ventral hippocampus of LC2-Hi, compared to LC2-Lo, rats is hard to interpret. Rewarding stimulation of the hippocampus has been described by several authors (2,6), and it is believed to be anatomically and functionally distinct from reward evoked by stimulation of the mesolimbic dopaminergic (lateral hypothalamic) system (2,6). Since the LC2 strains have been genetically separated according to lateral hypothalamic self-stimulation rates, no obvious predictions were made as to strain differences in hippocampal structures. It is, however, of interest to note that opioids are viewed as primary rewarding agents in the hippocampal system through binding to the ~ opioid receptor (30). This is in contrast to the modulatory role that opioids play in the mesolimbic-dopaminergic system (11). ACKNOWLEDGEMENT This study was supported in part by NIH Grant DA 05427.
REFERENCES 1. Belluzzi, J. D.; Stein, L. Enkephalin may mediate euphoria and drive-reduction reward. Nature 266:556-558; 1977. 2. Campbell, K. A.; Milgram, N. W. Mechanisms underlying the plasticity of hippocampal stimulation-induced reward. Behav. Neurosci. 99:209-219; 1985. 3. Carr, G. D.; Fibiger, H. C.; Phillips, A. G.; Conditioned place preference as a measure of drug reward. In: Liebman, J. M.; Cooper, S. J., eds. The pharmacological basis of reward. Oxford: Oxford University Press; 1989:264-319. 4. Cazala, P.; Cazals, Y.; Cardo, B. Hypothalamic self-stimulation in three inbred strains of mice. Brain Res. 81:159-167; 1974.
5. Cooper, S. J. Sweetness, reward and analgesia. Trends Pharmacol. Sci. 5:322-323; 1984. 6. Collier, T. J.; Routtenberg, A. Electrical self-stimulation of dentate gyrus granule ceils. Behav. Neural Biol. 42:85-90; 1984. 7. Esposito, R.; Kornetsky, C. Morphine lowering of self-stimulation thresholds: Lack of tolerance with long term administration. Science 195:189-191; 1977. 8. Ganchrow, J. R.; Lieblich, I.; Cohen, E. Consummatory responses to taste stimuli in rats selected for high and low rates of self-stimulation. Physiol. Behav. 27:971-976; 1981.
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9. Gross-lsseroff, R.; Dillon, K. A.; Israeli, M.; Biegon, A. Regionally selective increases in ~ opioid receptor density in the brains of suicide victims. Brain Res. 530:312-316; t990. 10. Koob, G. F.; Goeders, N. E. Neuroanatomical substrates of drug self-administration. In: Liebman, J. M.; Cooper, S. J., eds. The pharmacological basis of reward. Oxford: Oxford University Press; 1989: 214-263. 11. Leone, P.; Pocock, D.; Wise, R. A. Morphine-dopamine interaction: Ventral tegmental morphine increases nucleus accumbens dopamine release. Pharmacol. Biochem. Behav. 39:469-472; 1991. 12. Lieblich, I. Implications ofthe use ofthe genetic paradigm for brain research. In: Lieblich, I., ed. Genetics of the brain. Amsterdam: Elsevier; 1982:1-35. 13. Lieblich, 1. From a selection program on lateral hypothalamic selfstimulation to a neurochemical metaphor for hedonic and emotional behavior. In: Lieblich, 1., ed. Genetics of the brain. Amsterdam: Elsevier; 1982:231-270. 14. Lieblich, 1.; Cohen, E.; Beiles, A. Selection for high and for low rates of self-stimulation in rats. Physiol. Behav. 21:843-849: 1978. 15. Lieblich, I.; Cohen, E.; Ganchrow, J. R.; Blass, E. M.; Bergmann, F. Chronically elevated intake of saccharin solution induces morphine tolerance in genetically selected rats. Science 221:871-873: 1983. 16. Lieblich, 1.; Olds, J. Selection for the readiness to respond to electrical stimulation of the hypothalamus as a reinforcing agent. Brain Res. 27:153-161; 1971. 17. Lynch, W. C.; Libby, L. Naloxone suppresses intake of highly preferred saccarin solution in food deprived and sated rats. Life Sci. 33:1909-1914; 1983. 18. Mansur, R.; Khachaturian, H.; Lewis, M. E.: Akil, H.; Watson, S. G. Anatomy ofCNS opioid receptors. Trends Neurosci. 11:308314; 1988. 19. Marcus, R.; Kornetsky, C. Negative and positive intracranial reinforcement thresholds: Effects of morphine. Psychopharmacologia 38:1-13: 1974. 20. McLean, S.; Rothman, R. B.; Herkenham, M. Autoradiographic localization of u- and b-opiate receptors in the forebrain of the rat. Brain Res. 378:49-60; 1986. 21. Mucha, R. F.; Iversen, S. D. Reinforcing properties of morphine and naloxone revealed by conditioned place preference: A procedural examination. Psychopharmacology (Berlin) 82:241-247; 1984. 22. Nachman, M. The inheritance of saccharin preference. J. Comp. Physiol. Psychol. 3:73-138; 1959. 23. Olds, J.; Milner, P. Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J. Comp. Physiol. Psychol. 47:419-427; 1954. 24. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Sydney: Academic Press; 1986.
GROSS-ISSEROFF.
C O H E N AN[) S H A V I t
25. Phillips, A. G. Brain reward circuitD: A case for separate system~ Brain Res. Bull. 12:195-201:1984 26. Shavit, Y.: Cohen, E.; Steinberg, D.; Simantov, R. l)itterences oi opioid systems in rats genetically selected for high vs. low rates of self-stimulation. Pain 1989, Israel Pain Association, 2nd lntenlational Symposium. p. 30; 1989. 27. Shizgal, P.; Murray, B. Neuronal basis ofintracranial self-stimulation. In: Liebman, J. M.; Cooper, S, J., eds. The pharmacological basis of reward. Oxford: Oxford University Press; 1989:106- 163. 28. Siviy, S. M.; Calcagnetti, D. J.; Reid. L. D. Opioids and palatability. In: Hoebel, B. G.: Novin, D., eds. Neural basis of feeding and reward. Brunswick, ME: Haer Institute: 1982:517-524. 29. Stapleton, J. M.; Merriman, V. J.; Coogle, C. L.: (;elbard, S. D.: Reid, L. D. Naloxone reduces pressing for intracranial stimulation of sites in the priaqueductal gray area, accumbens nucleus, substantia nigra, and lateral hypothalamus. Physiol. Psychol. 7:427-436; 1970. 30. Stevens, K. E.; Shiotsu, G.; Stein, k. Hippocampal u-receptors mediate opioid reinforcement in the CA3 region. Brain Res. 545:8-16: 1991. 31. Tempel, A.; Zukin, R. S. Neuroanatomical patterns of the u, 6, and K opioid receptors of rat brain as determined by quantitative autoradiography. Proc. Natl. Acad. Sci. USA 84:4308-4312; 1987. 32. Trujillo, K. A.; Belluzzi, J. D.; Stein, L. Opiate antagonists and selfstimulation: Extinction-like response patterns suggest selective reward deficit. Brain Res. 492:15-28; 1989. 33. Vaccarino, F. J.; Bloom, F. E.; Koob, G. F. Blockade of nucleus accumbens opiate receptors attenuates intravenous heroin reward in the rat. Psychopharmacology (Berlin) 86:37-42; 1985. 34. Valenstein, E. S.; Lieblich, l.; Dinar, R.; Cohen, E.; Bachus, S. Relation between eating evoked by lateral hypothalamic stimulation and tail pinch in different rat strains. Behav. Neural Biol. 34:271282; 1982. 35. Van der Kooy, D.; Mucha, R. F.; O'Shaughnessy, M.; Bucenieks, P. Reinforcing effects of morphine revealed by conditioned place preference. Brain Res. 243:107-117: 1982. 36. West, T. E. G.; Wise, R. A. Effects of naltrexone on nucleus accumbens, lateral hypothalamic and ventral tegmental self-stimulation rate-frequency functions. Brain Res. 462:126-133; 1988. 37. Wise, R. A. Opiate reward: Sites and substrates. Neurosci. Biobehav. Rev. 13:129-133: 1989. 38. Wise, R. A.; Bozarth, M. A. Brain reward circuitry: Four circuit elements "'wired" in apparent series. Brain Res. Bull. 12:203-208; 1984. 39. Wise, R. A.; Raptis, L. Effects of naloxone and pimozide on initiation and maintenance measures of free feeding. Brain Res. 368:62-68; 1986.
0031-9384/92 $5.00+ .00 Copyright© 1992PergamonPress Ltd.
Printed in the USA.
BRIEF COMMUNICATION
Comparison of # Opioid Receptors in Brains of Rats Bred for High or Low Rate of Self-Stimulation R U T H G R O S S - I S S E R O F F , *~ E D N A COHEN~" A N D Y E H U D A S H A V I T t
*Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel and tDepartment of Psychology, The Hebrew University of Jerusalem, Jerusalem 91905, Israel Received 6 M a y 1991 GROSS-ISSEROFF, R., E. COHEN AND Y. SHAVIT. Comparisonof a opioid receptorsin brains of rats bredfor high or low rateofself-stimulation. PHYSIOL BEHAV 51(5) 1093-1096. 1992.--Opiates and endogenous opioid peptides play an important role in reward-mediated behaviors, including self-stimulation. Two strains of rats, LC2-Hi and LC2-Lo, selectivelybred for high vs. low rate of lateral hypothalamic self-stimulation, were employed in the present study. Quantitative autoradiography was performed on brains of adult male rats of each strain, using the a opioid receptor agonist 3H-DAGO. Strain differences in receptor density were observed in the nucleus accumbens and in ventral areas of the hippocampus. ~H-DAGO Autoradiography Nucleus accumbens
Self-stimulation
Genetic strains
ELECTRICAL stimulation of certain brain regions can be rewarding, as is evident from the phenomenon of self-stimulation (23). Intracranial self-stimulation (ICSS) is believed to involve the activation of central mechanisms which, under normal circumstances, subserve the processing of natural reward signals (23,25,27,38). Evidence from various studies, including studies of self-administration (10) and conditioned place preference (3,21,35), suggests that opiates and endogenous opioid peptides can also be rewarding [for a recent review see (37)]. It has been suggested that central reward mechanisms have genetic determinants (4,13,16,22). Several strains of rats, that differ in their response to lateral hypothalamic rewarding stimulation, have been selectively bred at the Hebrew University of Jerusalem (l 3,14). Two of these strains, LC2-Hi and LC2-Lo, were derived from a common ancestry and subjected to bidirectional selective pressure for high and low rates of ICSS, respectively. Consistent differences in rates of bar pressing for rewarding brain stimulation have been reported in these two strains (14). As demonstrated by further studies, other motivational differences evolved between these strains, concurrent with those related to ICSS. Compared with LC2-Lo, the LC2-Hi rats show more pronounced preference for sweet solutions (8), higher in-
~ opioid receptors
Reward
cidence of eating induced by lateral hypothalamic stimulation (34), and more robust morphine tolerance following prolonged maintenance on saccharin solution (15). In view of the possible involvement of endogenous opioids in reward associated with feeding (28,39), intake of sweet substances (5,15,17) and self-stimulation (1,7,19,29,32,36), it was hypothesized that the two strains might differ in the activity of their endogenous opioid systems. While there are obvious methodological difficulties in studying the correlation between motivational and other tendencies using a within-subject design, there is a clear advantage in using strain comparisons for this purpose [see (12,13)]. The differences previously established concerning the LC2 strains permit us to study endogenous opioid mechanisms in intact and experimentally naive rats whose ICSS characteristics are known (within the limits of a probabilistic estimation) without having been actually measured. Any difference found between these strains might be logically related to differences in their readiness to perform self-stimulation. Strain differences in the density of # opioid receptors were found in a preliminary saturation binding study, performed on homogenates of discrete brain areas taken from LC2-Hi and
Requests for reprints should be addressed to Ruth Isseroff, Biological Psychiatry Laboratory, Psychiatry Wing, The Chaim Sheba Medical Center, Tel Hashomer, Israel.
1093
1094
G R O S S - I S S E R O F F , (?OHEN A N D S H A V I I
TABLE 1 DISTRIBUTION OF tL OPIOID RECEPTORS IN LC2-HI AND LC2-LO RATS
Re,on
LC2-Hi [3H]DAGO bound
LC2-Lo
[~H]DAGO
LC2-t,o/
bound
LC2-Hi
309 _+ 65 [6] 381 ± 49 [6]
329 ± 31 [7] 363 ± 50 [7]
1.06 0.95
254 +_ 40 [7] 283 ± 57 [7]
236 ± 36 [7] 261 +_ 24 [7]
0.93 0.92
228 ± 31 [7] 246 _+ 34 [7]
195 +_ 28 [7] 191 _+ 38 [7]
0.86 0.78
218 219 204 197 897
[7] [7] [7] [7] [7]
0.95 1.04 0.86 0.76 0.85
476 ± 53 [7] 246-+ 29[7]
443 ± 60 [7] 2 1 7 ± 5t [7]
0.93 0.88
396 ± 51 [7] 271 ± 25 [7] 494 ± 102 [7]
382 ± 39 [7] 242 _+ 39 [7] 304 _+ 53 [7]
0.96 0,89 0.62
760 ± 70 [6] 609 ± 73 [6]
546 +_ 112 [6] 366 ± 82 [6] 518 310 352 957
:
~
t~
0.72 0.60
1.63 2.20
10 10
0.136 0.052
[6] [6] [6] [8]
0.68 0.52 0.50 0.81
2.16 3.30 2.23 1.7l
10 10 10 13
0.056 0.012 0.050 0.110
353 +_ 31 [7] 64 ± 5 [7] 645 -+ 80 [7]
455 _+ 41 [8] 84 ± 9 [7] 880 ± 43 [6]
1.29 1.31 1.36
1.89 1.97 2.45
13 12 11
0.082 0.072 0.032
592 -+ 89 [7] 188 -+ 22 [5] 208 -+ 47 [7]
390 ± 90 [8] 252 -+ 38 [8] 289 ± 104 [8]
0.65 1.34 1.38
480 _+ 124 [5] 434 ± 53 [6]
476 -+ 79 [7] 460 -+ 57 [6]
0.99 1.05
Cortex
Agranular insular Lateral orbital Frontal Area l Area 2 Somatomotor Forelimb area Hindlimb area Occipital Area l, binocular Area 1, monocular Area 2, lateral Area 2, mediolateral Presubiculum Hippocampus Dorsal CA fields Pyramidal layer Molecular layer Dentate gyrus Granular layer Molecular layer Hilus Ventral CA fields Pyramidal layer Molecular layer Dentate gyrus Granular layer Molecularlayer Hilus Amygdala Corpus striatum Caudate-putamen Globus pallidus Accumbens Thalamus Posterior nuclear group Ventrolateral nucleus Ventral posteromedial nucleus Hypothalamus Lateral Substantia Nigra
229 210 236 260 1061
_+ 34 _+ 36 ± 25 ± 21 ± 229
765 ± 594 -+ 706 ± 1180±
[6] [7] [6] [6] [6]
85 [6] 81 [6] 144 [6] 83 [7]
± ± ± +_ -+
-+ ± _+ ±
52 45 38 38 32
77 48 65 98
Table presents mean +_SEM specific [3H]DAGO binding in brains (number in brackets), expressed as fmol [3H]DAGO/mg protein, as determined by quantitative autoradiography. LC2-Lo/LC2-Hi--the proportion between the means of the two strains, t--value of t in a student's t-test; dr-degrees of freedom; p--probability of t in a two-way student's t-test. LC2-Lo rats. Bm~x values, obtained from Scatchard plots of tritiated D A G O b i n d i n g experiments, were significantly higher in the corpus striatum a n d thalamus of LC2-Lo brains, as compared with the respective regions o f LC2-Hi brains (p < 0.01, 2-tailed t-tests for m a t c h e d observations). No appreciable strain differences were observed in the hindbrain, midbrain, hypothalamus, h i p p o c a m p u s a n d anterior cerebral cortex (26). T h e present study was designed to confirm a n d extend these preliminary results. G i v e n the high resolution offered by the
quantitative autoradiography method, a n a t t e m p t was m a d e to p i n p o i n t differences in substructures within the gross regions excised for the h o m o g e n a t e experiments. The forebrain was chosen as a first target, in view of the previous findings. Eight LC2-Lo a n d 7 LC2-Hi adult male rats were used. Following decapitation brains were quickly removed, split at the midsagittal plane, frozen o n dry ice, a n d t h e n stored at - 7 0 ° C . T h e right cerebral hemisphere o f each a n i m a l was sectioned at - 1 5 ° C on a cryotome. Approximately 3 0 - 3 5 sagittal slices, 30
AUTORADIOGRAPHY OF # OPIOID RECEPTORS IN TWO RAT STRAINS #m thick, were obtained from each brain. Slices were thaw mounted on gelatin coated glass slides, kept overnight at - 2 0 ° C and then stored at - 7 0 ° C until use. Slides were incubated for 60 min at 25°C, with 1 nM 3H-DAGO (Tyr-D-Ala-Gly-Nmethyl-Phe-Gly-ol) (N.E.N., 58 Ci/mmol) in 50 mM Tris-HC1 buffer, pH 7.4 (9). Nonspecific binding was determined in the presence of 1 tiM etorphine. Slides were then washed for 9 min in the same ice cold buffer, dipped in ice cold water to remove buffer salts, dried on a 50°C hot plate and apposed to tritium sensitive film (Amersham 3H-Hyperfilm) for 12 weeks. The film was coexposed to commercial (Amersham) tritiated standards. It was manually developed and fixed with Kodak developer and fixer. Sections were stained with cresyl violet for anatomical reference; the Paxinos and Watson (24) rat brain atlas was used for identification of structures. Under the above conditions, nonspecific binding was at the level of film background. Autoradiograms were analyzed using an IBM-PC based computerized image analysis system with a PC-vision digitizing board (Imaging Technology, Inc.) and customized software. Standardization curves derived from the tritiated standards were used to convert gray level readings of the autoradiograms into equivalents of fmol 3H-DAGO/mg protein. Measurements of each region, within each brain, were done at least in duplicate. Mean values of 3H-DAGO binding, obtained from 30 brain regions and averaged within each strain, are presented in Table 1. As expected, we found a highly heterogeneous distribution of opioid receptors in the rat brain. High density of binding appeared over the amygdala, nucleus accumbens, and hypothalamus, whereas lower levels were found in the cortex, thalamus, and hippocampus. Heterogeneity was also observed within several of the brain regions studied: Receptor density was higher in the dorsal hippocampus than in the ventral hippocampus; receptors were more abundant in frontal areas of the cortex than in the occipital cortex; the caudate-putamen complex showed considerable binding, with a characteristic patchy pattern, while the globus pallidus had relatively low binding. The observed distribution was in good agreement with previously published results (20,31 ). Respective brain regions of the two strains were compared using 2-tailed t-tests; the results are presented in Table 1. In accordance with the results of the preliminary homogenate study, a higher 3H-DAGO binding was found in striatal structures of the LC2-Lo strain, as compared to the LC2-Hi respective structures. Significant strain differences in the predicted direction were observed in the nucleus accumbens. Similar differences were detected in the other striatal structures, the caudate-putamen and the globus pallidus; these differences, however, failed to reach statistical significance. Strain differences in the opposite direction were found in the ventral hippocampus. ~H-DAGO binding in the molecular layer
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and hilus of the ventral dentate gyrus were significantly higher in LC2-Hi than in LC2-Lo rats. A similar (but not significant) trend appeared in the granular layer of the ventral dentate gyrus and in the molecular layer of the ventral CA fields. No appreciable strain differences were detected in structures of the dorsal hippocampus. This might probably explain the absence of any indication of strain differences in assays of the entire hippocampal tissue in the previous study. Strain differences, which were previously observed in homogenates of the thalamus, were not confirmed by the present study. None of the other regions examined has shown significant strain differences in 3H-DAGO binding. The results of the autoradiography experiments do not allow to determine whether the differences in 3H-DAGO binding, observed between the two strains, are due to differences in affinity (Kd) or in U receptor density (Bmax), although the previous data of the saturation binding experiments (26) would favor the latter possibility. The most interesting finding reported here is the higher density of ~ opioid receptors in the nucleus accumbens of LC2-Lo compared with LC2-Hi rats. The nucleus accumbens is rich in opioid peptides (18) and opioid receptors (20,31); this structure has been associated with the mediation of the reinforcing effects ofopioids (33,36). Nucleus accumbens self-stimulation is attenuated by opiate antagonists (29,32,38). Based on findings of earlier studies, it has been suggested that the LC2-Lo strain is less hedonic and less responsive to incentive stimuli, compared with the LC2-Hi strain (8,13,15,34). The finding of higher density of opiate receptors in the nucleus accumbens of LC2-Lo rats might reflect lower activity of opioid peptides, or, paradoxically, an internal reward system which is more efficient and, therefore, less dependent on external (electrical, gustatory or other) stimulation. Further studies are needed to elucidate this point. The finding of higher ~ opioid receptor binding in the ventral hippocampus of LC2-Hi, compared to LC2-Lo, rats is hard to interpret. Rewarding stimulation of the hippocampus has been described by several authors (2,6), and it is believed to be anatomically and functionally distinct from reward evoked by stimulation of the mesolimbic dopaminergic (lateral hypothalamic) system (2,6). Since the LC2 strains have been genetically separated according to lateral hypothalamic self-stimulation rates, no obvious predictions were made as to strain differences in hippocampal structures. It is, however, of interest to note that opioids are viewed as primary rewarding agents in the hippocampal system through binding to the ~ opioid receptor (30). This is in contrast to the modulatory role that opioids play in the mesolimbic-dopaminergic system (11). ACKNOWLEDGEMENT This study was supported in part by NIH Grant DA 05427.
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