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Ontogeny of zeta (ζ), the opioid growth factor receptor, in the rat brain
Brain Research, 596 (1992) 149-156 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
149
BRES 18264
Ontogeny of zeta (st), the opioid growth factor receptor, in the rat brain Ian S. Zagon, Denise M. Gibo and Patricia J. McLaughlin Department of Neuroscience and Anatomy, The Pennsylvania State University Collegeof Medicine, The M.S. Hershey Medical Center, Hershey, PA 17033 (USA) (Accepted 16 June 1992)
Key words: Opioid receptor; Nervous system; Enkephalin; Growth; Development; Cerebellum; Brain; Zeta (~')receptor; Cancer; Mental retardation; Opioid antagonist; Naltrexone; Autism; Self-injurious behavior; Sudden infant death syndrome
Opioid growth factor (OGF), [Met5]enkephalin, serves as an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative events. OGF interacts with the zeta (g') opioid receptor to perform its function. Using [3H]-[MetS]enkephalin, the ontogeny of the g" receptor in the whole brain and cerebellum of rats was explored. Specific and saturable binding was recorded at the earliest time sampled, prenatal day 15 (El5). In the whole brain, binding capacity (Bmax) was two-fold greater at El5 than at El8 and E20. The quantity of g" receptor appeared to increase in the first postnatal week, reaching a maximum on postnatal day 8. Binding decreased the remainder of the 2nd week and between postnatal days 15 and 25 binding was no longer recorded. In the cerebellum, binding capacity increased from E20 to the 2nd postnatal week, reaching a maximum on postnatal days 8-10. The Bmax of the ~"receptor decreased precipitously on postnatal day 11, being 5.4-fold lower than on postnatal day 10. Between postnatal days 21 and 30, no binding was observed. The binding affinities of the whole brain and cerebellum were 2.3 and 2.7 nM, respectively, and no differences between ages could be detected. Continuous opioid receptor blockade from birth to postnatal day 6 increased body weight, the Bmax of the g" receptor in the whole brain and cerebellum (but not the gd), and increased the number of layers of germinal cells in the cerebellum. These results define the temporal expression of the g" receptor in the rat brain, as well as some regulatory properties, and support the concept that the ~"opioid receptor is primarily related to the proliferation of cells in the nervous system.
INTRODUCTION Growth factors are important in controlling cell
proliferation and differentiation during normal and abnormal development 15,19,29,43,45,54. Endogenous opio i d s 5'16'26'33'53'69'73, a s well as opioid receptors 3,4,6,7,10,16,21,26,33,36,39,44,51-53,57,5s, collectively known as endogenous opioid systems, are expressed during ontogeny and participate in the development of the nervous system 16'61-64'7~. Endogenous opioid peptides have been implicated in a number of crucial events associated with neuroembryology, including the proliferation, differentiation, and survival of neural cells 11'16'26'2s'61-64. An opioid growth factor (OGF), [MetS]enkephalin, has been identified in developing rat brain 7t, mouse neural tumor cells67, a wide variety of eukaryotic cells and tissues undergoing embryogenesis 16'73'74, and prokaryotes 7°. This naturally occurring pentapeptide is derived from proenkephalin A and serves as an inhibitory growth factor that is especially
targeted to cell proliferative events 64'67'7~, but also appears to be important in cellular differentiation and survival 62'63. Localization of OGF reveals an association with proliferating cells, as well as macroneurons, and the results are consistent with a temporal and spatial expression that is related to development 69'73. In situ hybridization studies showed that the source of this peptide appears to be both autocrine (from germinative neuroblasts) ~7 and paracrine (i.e. macroneurons) 17'29. Opioid action with respect to growth operates through classical opioid receptor mechanisms, being stereospecific and blocked by opioid antagonists (e.g. naloxone) 64'66'67m. Additionally, OGF has a tonic control on growth-related processes, since receptor blockade by potent opioid antagonists (i.e. naltrexone) elevates the number of cells undergoing DNA synthesis 64 and stimulates developmental events 61'63. The receptor related to OGF has been identified and characterized in the developing brain 58 and neural tumor cells 59, and termed the zeta (sr) opioid receptor.
Correspondence: I.S. Zagon, Department of Neuroscience and Anatomy, The M.S. Hershey Medical Center, Hershey, PA 17033, USA.
150 This receptor is present in developing but not adult human 5~ or rat ss brain. Binding to the receptor is stereospecific and displaceable by opioid antagonists, indicating compliance with the principles of opioid receptor involvement 35. The ~" opioid receptor is an integral membrane protein, proteinaceous in character and, unlike other opioid receptors 35, is associated with the nucleus 5s. Although the ~" receptor has been found to be important in governing the development of the nervous system, little is known about the ontogeny or regulation of this receptor in the mammalian brain. In the present study, receptor binding assays using radiolabeled [MetS]enkephalin were employed to formulate a profile of the g" receptor during brain and cerebellar development in rat. In addition, the effects of continuous opioid receptor blockade on the g" receptor were examined in order to begin exploring the properties of receptor activity.
s) in a 1 : 20 (w/v) solution of 50 mM Tris-HC! buffer with bacitracin {0.1 mg/ml), leupeptin (1 p.g/ml), thiorphan (6 riM), EGTA (1 raM), and phenylmethylsulfonyl fluoride (PMSF) (3.5 raM) ( = Tris/all buffer), pH 7.4, at 4°C, and stored (-70°C) until assayed (within 2 weeks). As described in earlier reports ss, the P1 pellet, a preparation enriched in nuclei 6°, was used in binding assays for both the whole brain and cerebellum. Homogenates were centrifuged at 2,200× g and the pellet resuspended in 0.32 M sucrose in Tris/all buffer. This was layered over a 1.4 M sucrose cushion and centrifuged at 2,200 × g; the procedure was performed twice. The pellet was resuspended in a Tris/all buffer (1:60, w / v ) a n d incubated at room temperature (22°C) for 20 rain to dissociate endogenous opioid peptides from receptors. Aliquots of tissue homogenate (0.95 ml) were incubated for 60 rain at 22°C with 50 #! of [3H]-[MetS]enkephalin; the final protein concentration was approximately 200/~g/ml. Incubation was terminated by rapid filtration through Whatman G F / B glass fiber filters under vacuum pressure with a Brandr.i Cell Harvester (Gaithersburg, MD). Filters were rinsed three times with 5 ml vols. of ice-cold 50 mM Tris-HC! buffer (pH 7.4), dried at 60°C for 1 h, and counted in a 2:1 solution of Aquasol-toluene by liquid scintillation spectror.tetry (Beckman LS-3801). Non-specific binding was determined ixJ the presence of excess (100 nM) unlabeled ligand. Homogenates in duplicate tubes were assayed for every concentration. Binding assays were replicated 2-8 times at each age examined. Protein concentrations were determined by the Bio-Rad assay with gamma globulin as a standard.
MATERIALS AND METHODS
Histolo;,y To examine the relationship of proliferating cells of the cerebellum to the ontogeny of the zeta receptor, prenatal and postnatal animals were anesthetized and perfused (intracardiac) with 10% net~tral buffered formalin. Cerebella were removed and processed for embedding in polyester wax. Midsagittal sections (8 Izm) were stained with hematoxylin and evaluated at 250x. The number of layers of cells comprising the external granule layer (EGL) on both sides of the primary fissure, midway between sulci and gyri, were recorded 6s. On prenatal day 20 and birth, cell counts were recorded in the middle of the anterior and posterior divisions of the cerebellar cortex. For each age, counts wore obtained from 3 sections/animal, 3 animals/age, and the mean number of cell layers was computed.
Animals and tissue preparation Male and female Sprague-Dawley rats (Charles River Labs., Wilmington, MA) were mated and their offspring used in this study. The presence of sperm in vaginal smears indicated gestation day 1. At birth, litters were culled to 8-10 pups per mother unless otherwise stated. All animals were housed under standard laboratory conditions as described elsewhere ~s. At designated times, animals were anesthetized, decapitated, and the entire brain or cerebellum removed. For embryonic tissues, time-mated females were anesthetized with nembutal, decapitated, embryos removed, and brain tissues dissected.
Receptor regulation
Receptor binding assay
To investigate the repercussions of opioid antagonist treatment on the ~' receptor, newborn rats were given a daily injection (i.p.) of naltrexon¢ hydrochloride (50 mg/kg) 61,62. Litters were culled to 10
Receptor binding assays were performed according to Zagon et al. 5s. Tissues were weighed and homogenized (Polytron, setting 5, 45
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ISHl-lMetSl-Enkephalln(nM) Fig. 1. Representative saturation binding isotherm and Scatchard plot (inset)of the specific binding of [3H]-[MetS]enkephalin to the P1 fraction of whole brain of 6-day-old rats. The average (mean + S.E.M.) K d generated from all assays of whole brain from 6-day-old animals was 2.4=1=0.5 nm and the Brnax was 10.6+ 1.8 fmol/mg protein.
151 pups per mother and one-half of the animals received naltrexone and one-half were given an injection of sterile water (vehicle). All animals were sacrificed on postnatal day 6, and the entire brain or cerebellum used in receptor binding assays under conditions described earlier. Binding assays for the whole brain and cerebellum were performed at least 2 times for each age examined.
Chemicals Reduced [3H]-[MetS]enkephalin (spec. act. 26.8 Ci/mmol) was obtained from DuPont-New England Nuclear Research Products (Boston, MA) as a custom-synthesized product. The following compounds were obtained from Sigma (St. Louis, MO): [MetSlenkephalin, naltrexone hydrochloride, leupeptin, bacitracin, EGTA, PMSF, and thiorphan.
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Statistical analysis Receptor binding data were analyzed with a Lundon I (Saturation Isotherm Binding Analysis) computer program (Lundon Software, Cleveland, OH). This analysis utilizes non-linear least-squares regression. Saturation curves and Scatchard plots were computed directly by this program. Data for binding affinity (K d) and binding capacity (Bmax) for each age were analyzed with a one-factor ANOVA. Data for body weight, B m a x values of whole brain and cerebellum, and the number of layers of EGL cells in the cerebellum, for naltrexone-treated and control animals were analyzed by ANOVA; subsequent planned comparisons were made using Newman-Keuls tests. RESULTS
Ontogeny of the zeta receptor in the rat brain Receptor binding assays of whole rat brain revealed specific and saturable binding of [3H]-[MetS]enkephalin in animals examined at prenatal days 15, 18, and 20, as well as at birth and postnatal days 1, 2, 3, 6, 8, 10, and 15 (Figs. 1, 2). A representative saturation and Scatchard plot of data are presented in Fig. 1. Although the binding capacities (Bmax) differed with each age examined, there was no difference in the binding affinities (Ko); the K o of all ages averaged 2.3:1:0.5
35 30
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P3
P6
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Age ( E m b r y o n i c a n d P o s t n a t a l Days)
Fig. 2. Specific and saturable binding of [3Hl-[MetSlenkephalin to the P1 fraction of whole brains of prenatal (El5, El8, E20 days), newborn (P0), postnatal (P) (1, 2, 3, 6, 8, 10, 15, 25, 35 days) and adult (AD) rats. Data are expressed as mean + S.E.M. * -- no specific and saturable binding.
Fig. 3. Specific and saturable binding of [3Hl-[MetS]enkephalin to the P1 fraction of the cerebellum from rats of prenatal (day E20), newborn (P0), postnatal (P) (1, 2, 3, 6, 8, 10, 11, 12, 15, 21, 30, 35 days) and adult (AD). Data are expressed as mean :1:S.E.M. * -- no specific and saturable binding.
nM. It appears that early in the embryology of the brain (i.e. prenatal day 15), there is two-fold more ~" receptors than in subsequent prenatal periods (i.e. prenatal days 18 and 20). The quantity of ~" receptor appears to rise in the first postnatal week, reaching a peak on postnatal day 8. In the remainder of the 2nd week, and continuing into the 3rd week, binding capacities for radiolabeled [MetS]enkephalin binding decreased. Between postnatal days 15 and 25 and continuing into adulthood, specific and saturable binding was no longer recorded in the whole rat brain. Ontogeny of the zeta receptor in the cerebellum Receptor binding assays of the rat cerebellum revealed specific and saturable binding of [3H]-[MetS]enkephalin in animals examined at prenatal day 20, as well as at birth and postnatal days 1, 2, 3, 6, 8, 10, 11, 12, 15, and 21 (Fig. 3). Although the binding capacities (Bma x) differed with each age examined, there was no difference in the binding affinities (Kd); the K,~ of all ages averaged 2.7 + 0.5 nM. Binding was recorded at prenatal day 20; difficulty in delineating the cerebellum and obtaining sufficient quantities of tissue prohibited assessment of receptor binding at earlier ages. Between birth and day 3, a greater than 3-fold increase in binding was noted, and there was 5 times more binding in the cerebella of postnatal day 10 animals than neonates. A sharp decrease in binding was observed between postnatal days 10 and 11. No marked differences in receptor binding could be detected from postnatal day 11 to weaning. Between postnatal days 21 and 30, and continuing into adulthood, specific and saturable binding was no longer recorded in the cerebellum.
152
Ontogeny of the EGL
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The ontogeny of the EGL in the rat cerebellum was evaluated in the primary fissure from prenatal day 20 (E20) through the preweaning period (Fig. 4). The sum total of the superficial and deep layers of the EGL (EGL-S and EGL-D according to Zagon and MeLaughlin6s) was assessed. The EGL was 2-3 cell layers deep shortly before birth (i.e. E20), and increased approximately 3-fold between birth and postnatal day 6. The EGL appeared to slowly decrease in depth between postnatal days 6 and 10, and by postnatal day 12 the EGL was approximately 40% less than recorded at postnatal day 6. At postnatal day 15, the EGL had decreased 3.5-fold from postnatal day 6 and by weaning (postnatal day 21) the EGL was either no longer present or consisted of 1 cell layer; beyond weaning the EGL was not observed. Although the EGL-S and EGL-D were not analyzed separately, qualitatively the EGL-S reached its optimum between postnatal days 6 and 8, and noticeably diminished by the 3rd postnatal week. The EGL-D was most prominent between postnatal days 6 and 10, and also declined in the 3rd postnatal week.
Receptor regulation The influence of continuous opioid receptor blockade on the K d and Bma x of the ~" receptor were evaluated. Animals receiving daily in]ections of 50 mg/kg naltrexone, a regimen known to continuously block the antinociceptive effects of an opioid agonist 6t'°2, were examined on postnatal day 6 (Fig. 5). Animals subjected to a continuous receptor blockade showed a significant increase (14%) in body weight from control values (12.2 :l: 0.3 g). The Bmax of [3H][Met5]enkephalin in the whole brain and cerebe~iual
I0
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P2
P4
P0
P8
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P15
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Age ( E m b r y o n i c a n d P o s t n a t a l Days)
Fig. 4. The number of layers of EGL cells in the primary fissure of the rat cerebellum from animals of prenatal (E20), newborn (P0), and postnatal (P) (2, 3, 6, 8, 10, 12, 15, 18, 21) days. At E20 and birth, the number of layers of EGL cells was determined from a point midway between the anterior and posterior divisions of the vermis. Data are expressed as mean + S.E.M.
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Fig. 5. The influence of continuous opioid receptor blockade on the body weight, binding (Bmax) of [3H]-[MetS]enkephalin in the P1 fraction of the whole brain and cerebellum, and the number of cell layers comprising the EGL of the cerebellum in the 6-day-old rat. Significantly different from control values at P < 0.05 (*) or P < 0.01 (* *). Bars = S.E.M.
were approximately 45% and 66% greater than in control subjects (22.9 + 1.4 and 27.8 + 3.6 fmol/mg protein, respectively); in both cases, differences from controls were statistically reliable. No change in the K d was obseived. Histological analysis of the EGL cell depth in naltrexone-treated rats revealed a significant increase (20%) from control levels (9.0 + 0.4), with a range of 10-13 layers recorded in the naltrexone group in contrast to a range of 8-10 layers in the control group. DISCUSSION [MetS]enkephalin is an OGF that modulates the growth and proliferation of normal 7~ and abnormaP 7 cOls and tissues in a stereospecific and opioid antagonist-reversible fashion. The receptor responsible for mediating OGF activity has been identified as the ~' opioid receptor 5s,sg. In the present study we have found that the ~" receptor is present in both the whole brain and the cerebellum during morphogenesis, but not in the nervous system of adult rats, confirming an earlier report utilizing the cerebellum of 6-day-old and adult rats 5s. It is now clear that this receptor can be ,[~tected in the prenatal period, a time when the nervous system is just beginning to be formed in the rat. Moreover, the data indicate that the ~" receptor is present in the developing nervous system during a well-defined period, occurring from prenatal day 15 (the earliest point sampled) up to weaning (the end of the 3rd postnatal week). By weaning, neurogenesis is completed (see Jacobsen Is) and the ~"receptor can no longer be found. Our data also show that blockade of the ~"receptor has considerable repercussions on the number, but not
153 binding affinity, of this receptor. We found that the concentration of ~" receptor is increased in both the whole brain and the cerebellum during receptor blockade, along with a corresponding increase in the number of cell layers composing the EGL. Thus, these results are the first to report the timetable of g" receptor ontogeny in the nervous system, the presence of the receptor in the entire brain, and information about the regulation of this receptor by opioid receptor perturbation. The ~" receptor in the developing nervous system of rats appears to be related to proliferating neural cells. A number of observations lead us to this conclusion. First, the development of the nervous system takes considerable time in the rat (see Jacobson ~s) and consists of cell proliferation, migration, and differentiation which may occur sequentially or in an overlapping fashion. The ~" receptor appears to be correlated only with the earliest phases of neural ontogeny, a time characterized by active cell proliferation. In the cerebellum the relationship between the presence of the EGL cells, a germinative matrix that gives rise to microneurons ~, and the ontogenic profile of the receptor is most easily perceived. In the whole brain, cell proliferation e~curs in the prenatal period and in certain brain regions (e.g., hippocampus) during the first 3 weeks after birth (see JacobsonlS). After weaning, a period characterized by nervous system maturation and differentiation, the g" receptor is not present. Although
OGF regulates glial proliferation, and gliogenesis takes place largely in the postweaning period (see Jacobson~S), the absence of g" receptor binding after postnatal day 21 may reflect a lack of sensitivity by the
binding assay to detect receptors associated with replicating glia; in vitro autoradiography and the use of g" receptor antibodies will be needed to address this
point. Second, autoradiography experiments using [3H]thymidine16'64m have documented that OGF controls cell proliferation in the developing nervous system. Therefore, mediation of OGF action on cell replication by the ~" receptor would be expected to occur during the period when there is receptor binding activity. Third, in vitro autoradiography investigations in the retina have shown that [MetS]enkephalin binding was associated with the germinal neuroblast layer of the neonate in a naloxone-reversible manner, and not in the adult retina ~6. These data would suggest that proliferating cells have a distinct relationship with the OGF receptor. Fourth, earlier studies 16'69'73 have shown that OGF is found in developing but not adult neural tissues, and is prominent in proliferating cells destined to be neurons and glia. It might be expected that the ~" receptor would be localized with OGF-containing pro-
liferative cells. Fifth, the ~" receptor has been identified in neural tumor cells in tissue culture 59, indicating the relationship of proliferating neural cells to this receptor. Studies now in progress using receptor ligand autoradiography and antibodies to the ~" receptor should be capable of elucidating the location of the sr receptor and complement the receptor binding data in this report. An intriguing observation recorded in the present investigation was the marked decrease in the number of ~" receptors in the cerebellum between postnatal days 10 and 11. Within a 24-h period, a 5.4-fold decrease in receptors was noted. However, the decrease in the number of layers of EGL cells in the cerebellum of .animals of postnatal days 10-12 was only about 25%. The reason(s) for the notable decrease in ~" receptors on day 11, despite a relatively small change in the number of cells comprising the EGL, is unclear. Altman ~, examining the changes in the depth of the EGL of the pyram!s in the developing cerebellum of the rat, noted that "From the eleventh day onward there is a sharp decline in the depth of the proliferative zone .... " (p. 357). If ~" receptors play a role in governing cell proliferation, it would be understandable that a decrease in the number of dividing cells would correlate with a reduction in the number of these receptors. Earlier investigations have shown that opioid receptor blockade has a profound effect on the developing organism 11,16,61-64. It is known that opioid receptor blockade often results in an up-regulation of opioid receptors, opioid gene expression, and opioid peptides 3,8,37,47-51,55,6s, and enhances physiological response to opioids 22'41'46'55'56'6s. In the present study we show that continuous opioid receptor blockade in developing animals results in an increase in body weight, confirming earlier findings6~'62'66. This regimen of receptor blockade also produces an increase in the number of g" receptors in the whole brain and in the cerebellum. Apparently, an increase in ~" receptors is a compensation for blockade of these opioid receptors. Thus, even though the ~" receptor differs in function and subcellular distribution from other opioid receptors, this receptor shares a number of properties with other opioid receptors (e.g~ ~, 8, K) such as an up-regulatory response to opioid receptor blockade, stereospecifieity, and affinity for opioid antagonists. It is also of interest to note that opioid receptor blockade results in an increase in DNA synthesis occurring in germinal neural cells64. The present data indicate that at least in one germinative matrix, the EGL of the cerebellum, the number of layers of EGL cells is substantially increased from control levels in the ccrebel-
154
lar cortex of 6-day-old rats given a continuous opioid receptor blockade. This information is consistent with previous findings that the number of neurons (and gila) are significantly increased in the brains of animals given a continuous opioid receptor blockade throughout the preweaning period 6.1'62. Our findings with respect to the ontogeny of the ~" receptor in the developing rat brain may have considerable relevance to the human situation. [MetS]enkepha lin binding sites have been identified in homogenates of human cerebellar tissue 57, with binding observed at birth and being greatest during infancy; no specific and saturable binding of radiolabeled [Met5]enkephalin was recorded in the cerebellum of adult humans. We have postulated ~1,27,58.71 that alterations in endogenous opiold systems may be related to the etiology and pathogenesis of some clinical disorders. For example, studies have reported an association between opioids and autism 13'20'24'25'27'42, self-injurious behavior 12,14, sudden infant death syndrome 3'.3s, and cancer 59'66'6s. Given the results of the present investigations, it could be hypothesized that disturbances in the ontogeny of the ~" receptor results in dysgenesis of the nervous system. Thus, an insufficient number of ~" receptors, a reduction in the affinity of this receptor, and/or a decrease in OGF might be expected to produce an increased number of cells and/or stimulation in the development of brain circuitry. This has already been documented by experiments with continuous opioid receptor blockade in the rat II,6~-63.Depending on the degree of br receptor dysfunction, neoplasia of the nervous system, the second most common cancer in children (under 15) 2, may even occur. Indeed, a strong relationship between neural cancer and [MetS]enkephalin/~ receptor has already been established 59,6z6s,72. Furthermore, an increase in the number of ~" receptors, a greater affinity of this receptor, and/or overabundance of OGF might be expected to result in a decrease in the number of cells and/or retardation in development of dendrites and synapses. Once again, depending on the severity of receptor dysfunction, impairment in the number of cells and ultimately the circuitry of the brain could result. Research on the effects of intermittent opioid receptor blockade ~1,62,63 support this contention, presumably with an opioid antagonist provoked excess of OGF interacting with an increased number of ~" receptors during the interval when the opioid antagonist is no longer available. These clinical implications only emphasize the importance of understanding how OGF and the ~" receptor participate in governing the development of the nervous system.
Acknowledgements. This work was supported by NIH Grant NS20500. We thank Dr. Tomoki Isayama for critical evaluation of the manuscript and his assistance in preparing some of the illustrations.
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149
BRES 18264
Ontogeny of zeta (st), the opioid growth factor receptor, in the rat brain Ian S. Zagon, Denise M. Gibo and Patricia J. McLaughlin Department of Neuroscience and Anatomy, The Pennsylvania State University Collegeof Medicine, The M.S. Hershey Medical Center, Hershey, PA 17033 (USA) (Accepted 16 June 1992)
Key words: Opioid receptor; Nervous system; Enkephalin; Growth; Development; Cerebellum; Brain; Zeta (~')receptor; Cancer; Mental retardation; Opioid antagonist; Naltrexone; Autism; Self-injurious behavior; Sudden infant death syndrome
Opioid growth factor (OGF), [Met5]enkephalin, serves as an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative events. OGF interacts with the zeta (g') opioid receptor to perform its function. Using [3H]-[MetS]enkephalin, the ontogeny of the g" receptor in the whole brain and cerebellum of rats was explored. Specific and saturable binding was recorded at the earliest time sampled, prenatal day 15 (El5). In the whole brain, binding capacity (Bmax) was two-fold greater at El5 than at El8 and E20. The quantity of g" receptor appeared to increase in the first postnatal week, reaching a maximum on postnatal day 8. Binding decreased the remainder of the 2nd week and between postnatal days 15 and 25 binding was no longer recorded. In the cerebellum, binding capacity increased from E20 to the 2nd postnatal week, reaching a maximum on postnatal days 8-10. The Bmax of the ~"receptor decreased precipitously on postnatal day 11, being 5.4-fold lower than on postnatal day 10. Between postnatal days 21 and 30, no binding was observed. The binding affinities of the whole brain and cerebellum were 2.3 and 2.7 nM, respectively, and no differences between ages could be detected. Continuous opioid receptor blockade from birth to postnatal day 6 increased body weight, the Bmax of the g" receptor in the whole brain and cerebellum (but not the gd), and increased the number of layers of germinal cells in the cerebellum. These results define the temporal expression of the g" receptor in the rat brain, as well as some regulatory properties, and support the concept that the ~"opioid receptor is primarily related to the proliferation of cells in the nervous system.
INTRODUCTION Growth factors are important in controlling cell
proliferation and differentiation during normal and abnormal development 15,19,29,43,45,54. Endogenous opio i d s 5'16'26'33'53'69'73, a s well as opioid receptors 3,4,6,7,10,16,21,26,33,36,39,44,51-53,57,5s, collectively known as endogenous opioid systems, are expressed during ontogeny and participate in the development of the nervous system 16'61-64'7~. Endogenous opioid peptides have been implicated in a number of crucial events associated with neuroembryology, including the proliferation, differentiation, and survival of neural cells 11'16'26'2s'61-64. An opioid growth factor (OGF), [MetS]enkephalin, has been identified in developing rat brain 7t, mouse neural tumor cells67, a wide variety of eukaryotic cells and tissues undergoing embryogenesis 16'73'74, and prokaryotes 7°. This naturally occurring pentapeptide is derived from proenkephalin A and serves as an inhibitory growth factor that is especially
targeted to cell proliferative events 64'67'7~, but also appears to be important in cellular differentiation and survival 62'63. Localization of OGF reveals an association with proliferating cells, as well as macroneurons, and the results are consistent with a temporal and spatial expression that is related to development 69'73. In situ hybridization studies showed that the source of this peptide appears to be both autocrine (from germinative neuroblasts) ~7 and paracrine (i.e. macroneurons) 17'29. Opioid action with respect to growth operates through classical opioid receptor mechanisms, being stereospecific and blocked by opioid antagonists (e.g. naloxone) 64'66'67m. Additionally, OGF has a tonic control on growth-related processes, since receptor blockade by potent opioid antagonists (i.e. naltrexone) elevates the number of cells undergoing DNA synthesis 64 and stimulates developmental events 61'63. The receptor related to OGF has been identified and characterized in the developing brain 58 and neural tumor cells 59, and termed the zeta (sr) opioid receptor.
Correspondence: I.S. Zagon, Department of Neuroscience and Anatomy, The M.S. Hershey Medical Center, Hershey, PA 17033, USA.
150 This receptor is present in developing but not adult human 5~ or rat ss brain. Binding to the receptor is stereospecific and displaceable by opioid antagonists, indicating compliance with the principles of opioid receptor involvement 35. The ~" opioid receptor is an integral membrane protein, proteinaceous in character and, unlike other opioid receptors 35, is associated with the nucleus 5s. Although the ~" receptor has been found to be important in governing the development of the nervous system, little is known about the ontogeny or regulation of this receptor in the mammalian brain. In the present study, receptor binding assays using radiolabeled [MetS]enkephalin were employed to formulate a profile of the g" receptor during brain and cerebellar development in rat. In addition, the effects of continuous opioid receptor blockade on the g" receptor were examined in order to begin exploring the properties of receptor activity.
s) in a 1 : 20 (w/v) solution of 50 mM Tris-HC! buffer with bacitracin {0.1 mg/ml), leupeptin (1 p.g/ml), thiorphan (6 riM), EGTA (1 raM), and phenylmethylsulfonyl fluoride (PMSF) (3.5 raM) ( = Tris/all buffer), pH 7.4, at 4°C, and stored (-70°C) until assayed (within 2 weeks). As described in earlier reports ss, the P1 pellet, a preparation enriched in nuclei 6°, was used in binding assays for both the whole brain and cerebellum. Homogenates were centrifuged at 2,200× g and the pellet resuspended in 0.32 M sucrose in Tris/all buffer. This was layered over a 1.4 M sucrose cushion and centrifuged at 2,200 × g; the procedure was performed twice. The pellet was resuspended in a Tris/all buffer (1:60, w / v ) a n d incubated at room temperature (22°C) for 20 rain to dissociate endogenous opioid peptides from receptors. Aliquots of tissue homogenate (0.95 ml) were incubated for 60 rain at 22°C with 50 #! of [3H]-[MetS]enkephalin; the final protein concentration was approximately 200/~g/ml. Incubation was terminated by rapid filtration through Whatman G F / B glass fiber filters under vacuum pressure with a Brandr.i Cell Harvester (Gaithersburg, MD). Filters were rinsed three times with 5 ml vols. of ice-cold 50 mM Tris-HC! buffer (pH 7.4), dried at 60°C for 1 h, and counted in a 2:1 solution of Aquasol-toluene by liquid scintillation spectror.tetry (Beckman LS-3801). Non-specific binding was determined ixJ the presence of excess (100 nM) unlabeled ligand. Homogenates in duplicate tubes were assayed for every concentration. Binding assays were replicated 2-8 times at each age examined. Protein concentrations were determined by the Bio-Rad assay with gamma globulin as a standard.
MATERIALS AND METHODS
Histolo;,y To examine the relationship of proliferating cells of the cerebellum to the ontogeny of the zeta receptor, prenatal and postnatal animals were anesthetized and perfused (intracardiac) with 10% net~tral buffered formalin. Cerebella were removed and processed for embedding in polyester wax. Midsagittal sections (8 Izm) were stained with hematoxylin and evaluated at 250x. The number of layers of cells comprising the external granule layer (EGL) on both sides of the primary fissure, midway between sulci and gyri, were recorded 6s. On prenatal day 20 and birth, cell counts were recorded in the middle of the anterior and posterior divisions of the cerebellar cortex. For each age, counts wore obtained from 3 sections/animal, 3 animals/age, and the mean number of cell layers was computed.
Animals and tissue preparation Male and female Sprague-Dawley rats (Charles River Labs., Wilmington, MA) were mated and their offspring used in this study. The presence of sperm in vaginal smears indicated gestation day 1. At birth, litters were culled to 8-10 pups per mother unless otherwise stated. All animals were housed under standard laboratory conditions as described elsewhere ~s. At designated times, animals were anesthetized, decapitated, and the entire brain or cerebellum removed. For embryonic tissues, time-mated females were anesthetized with nembutal, decapitated, embryos removed, and brain tissues dissected.
Receptor regulation
Receptor binding assay
To investigate the repercussions of opioid antagonist treatment on the ~' receptor, newborn rats were given a daily injection (i.p.) of naltrexon¢ hydrochloride (50 mg/kg) 61,62. Litters were culled to 10
Receptor binding assays were performed according to Zagon et al. 5s. Tissues were weighed and homogenized (Polytron, setting 5, 45
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ISHl-lMetSl-Enkephalln(nM) Fig. 1. Representative saturation binding isotherm and Scatchard plot (inset)of the specific binding of [3H]-[MetS]enkephalin to the P1 fraction of whole brain of 6-day-old rats. The average (mean + S.E.M.) K d generated from all assays of whole brain from 6-day-old animals was 2.4=1=0.5 nm and the Brnax was 10.6+ 1.8 fmol/mg protein.
151 pups per mother and one-half of the animals received naltrexone and one-half were given an injection of sterile water (vehicle). All animals were sacrificed on postnatal day 6, and the entire brain or cerebellum used in receptor binding assays under conditions described earlier. Binding assays for the whole brain and cerebellum were performed at least 2 times for each age examined.
Chemicals Reduced [3H]-[MetS]enkephalin (spec. act. 26.8 Ci/mmol) was obtained from DuPont-New England Nuclear Research Products (Boston, MA) as a custom-synthesized product. The following compounds were obtained from Sigma (St. Louis, MO): [MetSlenkephalin, naltrexone hydrochloride, leupeptin, bacitracin, EGTA, PMSF, and thiorphan.
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Statistical analysis Receptor binding data were analyzed with a Lundon I (Saturation Isotherm Binding Analysis) computer program (Lundon Software, Cleveland, OH). This analysis utilizes non-linear least-squares regression. Saturation curves and Scatchard plots were computed directly by this program. Data for binding affinity (K d) and binding capacity (Bmax) for each age were analyzed with a one-factor ANOVA. Data for body weight, B m a x values of whole brain and cerebellum, and the number of layers of EGL cells in the cerebellum, for naltrexone-treated and control animals were analyzed by ANOVA; subsequent planned comparisons were made using Newman-Keuls tests. RESULTS
Ontogeny of the zeta receptor in the rat brain Receptor binding assays of whole rat brain revealed specific and saturable binding of [3H]-[MetS]enkephalin in animals examined at prenatal days 15, 18, and 20, as well as at birth and postnatal days 1, 2, 3, 6, 8, 10, and 15 (Figs. 1, 2). A representative saturation and Scatchard plot of data are presented in Fig. 1. Although the binding capacities (Bmax) differed with each age examined, there was no difference in the binding affinities (Ko); the K o of all ages averaged 2.3:1:0.5
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Fig. 2. Specific and saturable binding of [3Hl-[MetSlenkephalin to the P1 fraction of whole brains of prenatal (El5, El8, E20 days), newborn (P0), postnatal (P) (1, 2, 3, 6, 8, 10, 15, 25, 35 days) and adult (AD) rats. Data are expressed as mean + S.E.M. * -- no specific and saturable binding.
Fig. 3. Specific and saturable binding of [3Hl-[MetS]enkephalin to the P1 fraction of the cerebellum from rats of prenatal (day E20), newborn (P0), postnatal (P) (1, 2, 3, 6, 8, 10, 11, 12, 15, 21, 30, 35 days) and adult (AD). Data are expressed as mean :1:S.E.M. * -- no specific and saturable binding.
nM. It appears that early in the embryology of the brain (i.e. prenatal day 15), there is two-fold more ~" receptors than in subsequent prenatal periods (i.e. prenatal days 18 and 20). The quantity of ~" receptor appears to rise in the first postnatal week, reaching a peak on postnatal day 8. In the remainder of the 2nd week, and continuing into the 3rd week, binding capacities for radiolabeled [MetS]enkephalin binding decreased. Between postnatal days 15 and 25 and continuing into adulthood, specific and saturable binding was no longer recorded in the whole rat brain. Ontogeny of the zeta receptor in the cerebellum Receptor binding assays of the rat cerebellum revealed specific and saturable binding of [3H]-[MetS]enkephalin in animals examined at prenatal day 20, as well as at birth and postnatal days 1, 2, 3, 6, 8, 10, 11, 12, 15, and 21 (Fig. 3). Although the binding capacities (Bma x) differed with each age examined, there was no difference in the binding affinities (Kd); the K,~ of all ages averaged 2.7 + 0.5 nM. Binding was recorded at prenatal day 20; difficulty in delineating the cerebellum and obtaining sufficient quantities of tissue prohibited assessment of receptor binding at earlier ages. Between birth and day 3, a greater than 3-fold increase in binding was noted, and there was 5 times more binding in the cerebella of postnatal day 10 animals than neonates. A sharp decrease in binding was observed between postnatal days 10 and 11. No marked differences in receptor binding could be detected from postnatal day 11 to weaning. Between postnatal days 21 and 30, and continuing into adulthood, specific and saturable binding was no longer recorded in the cerebellum.
152
Ontogeny of the EGL
12o
The ontogeny of the EGL in the rat cerebellum was evaluated in the primary fissure from prenatal day 20 (E20) through the preweaning period (Fig. 4). The sum total of the superficial and deep layers of the EGL (EGL-S and EGL-D according to Zagon and MeLaughlin6s) was assessed. The EGL was 2-3 cell layers deep shortly before birth (i.e. E20), and increased approximately 3-fold between birth and postnatal day 6. The EGL appeared to slowly decrease in depth between postnatal days 6 and 10, and by postnatal day 12 the EGL was approximately 40% less than recorded at postnatal day 6. At postnatal day 15, the EGL had decreased 3.5-fold from postnatal day 6 and by weaning (postnatal day 21) the EGL was either no longer present or consisted of 1 cell layer; beyond weaning the EGL was not observed. Although the EGL-S and EGL-D were not analyzed separately, qualitatively the EGL-S reached its optimum between postnatal days 6 and 8, and noticeably diminished by the 3rd postnatal week. The EGL-D was most prominent between postnatal days 6 and 10, and also declined in the 3rd postnatal week.
Receptor regulation The influence of continuous opioid receptor blockade on the K d and Bma x of the ~" receptor were evaluated. Animals receiving daily in]ections of 50 mg/kg naltrexone, a regimen known to continuously block the antinociceptive effects of an opioid agonist 6t'°2, were examined on postnatal day 6 (Fig. 5). Animals subjected to a continuous receptor blockade showed a significant increase (14%) in body weight from control values (12.2 :l: 0.3 g). The Bmax of [3H][Met5]enkephalin in the whole brain and cerebe~iual
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PI8
P21
Age ( E m b r y o n i c a n d P o s t n a t a l Days)
Fig. 4. The number of layers of EGL cells in the primary fissure of the rat cerebellum from animals of prenatal (E20), newborn (P0), and postnatal (P) (2, 3, 6, 8, 10, 12, 15, 18, 21) days. At E20 and birth, the number of layers of EGL cells was determined from a point midway between the anterior and posterior divisions of the vermis. Data are expressed as mean + S.E.M.
* :i:
G
O
o k
100
80
:t: t.
--
40
~
20
/
/
0 Body Weight
Whole B r a i n ¢ Receptor
Cerebellum ¢ Receptor
EGL Cell Depth
Fig. 5. The influence of continuous opioid receptor blockade on the body weight, binding (Bmax) of [3H]-[MetS]enkephalin in the P1 fraction of the whole brain and cerebellum, and the number of cell layers comprising the EGL of the cerebellum in the 6-day-old rat. Significantly different from control values at P < 0.05 (*) or P < 0.01 (* *). Bars = S.E.M.
were approximately 45% and 66% greater than in control subjects (22.9 + 1.4 and 27.8 + 3.6 fmol/mg protein, respectively); in both cases, differences from controls were statistically reliable. No change in the K d was obseived. Histological analysis of the EGL cell depth in naltrexone-treated rats revealed a significant increase (20%) from control levels (9.0 + 0.4), with a range of 10-13 layers recorded in the naltrexone group in contrast to a range of 8-10 layers in the control group. DISCUSSION [MetS]enkephalin is an OGF that modulates the growth and proliferation of normal 7~ and abnormaP 7 cOls and tissues in a stereospecific and opioid antagonist-reversible fashion. The receptor responsible for mediating OGF activity has been identified as the ~' opioid receptor 5s,sg. In the present study we have found that the ~" receptor is present in both the whole brain and the cerebellum during morphogenesis, but not in the nervous system of adult rats, confirming an earlier report utilizing the cerebellum of 6-day-old and adult rats 5s. It is now clear that this receptor can be ,[~tected in the prenatal period, a time when the nervous system is just beginning to be formed in the rat. Moreover, the data indicate that the ~" receptor is present in the developing nervous system during a well-defined period, occurring from prenatal day 15 (the earliest point sampled) up to weaning (the end of the 3rd postnatal week). By weaning, neurogenesis is completed (see Jacobsen Is) and the ~"receptor can no longer be found. Our data also show that blockade of the ~"receptor has considerable repercussions on the number, but not
153 binding affinity, of this receptor. We found that the concentration of ~" receptor is increased in both the whole brain and the cerebellum during receptor blockade, along with a corresponding increase in the number of cell layers composing the EGL. Thus, these results are the first to report the timetable of g" receptor ontogeny in the nervous system, the presence of the receptor in the entire brain, and information about the regulation of this receptor by opioid receptor perturbation. The ~" receptor in the developing nervous system of rats appears to be related to proliferating neural cells. A number of observations lead us to this conclusion. First, the development of the nervous system takes considerable time in the rat (see Jacobson ~s) and consists of cell proliferation, migration, and differentiation which may occur sequentially or in an overlapping fashion. The ~" receptor appears to be correlated only with the earliest phases of neural ontogeny, a time characterized by active cell proliferation. In the cerebellum the relationship between the presence of the EGL cells, a germinative matrix that gives rise to microneurons ~, and the ontogenic profile of the receptor is most easily perceived. In the whole brain, cell proliferation e~curs in the prenatal period and in certain brain regions (e.g., hippocampus) during the first 3 weeks after birth (see JacobsonlS). After weaning, a period characterized by nervous system maturation and differentiation, the g" receptor is not present. Although
OGF regulates glial proliferation, and gliogenesis takes place largely in the postweaning period (see Jacobson~S), the absence of g" receptor binding after postnatal day 21 may reflect a lack of sensitivity by the
binding assay to detect receptors associated with replicating glia; in vitro autoradiography and the use of g" receptor antibodies will be needed to address this
point. Second, autoradiography experiments using [3H]thymidine16'64m have documented that OGF controls cell proliferation in the developing nervous system. Therefore, mediation of OGF action on cell replication by the ~" receptor would be expected to occur during the period when there is receptor binding activity. Third, in vitro autoradiography investigations in the retina have shown that [MetS]enkephalin binding was associated with the germinal neuroblast layer of the neonate in a naloxone-reversible manner, and not in the adult retina ~6. These data would suggest that proliferating cells have a distinct relationship with the OGF receptor. Fourth, earlier studies 16'69'73 have shown that OGF is found in developing but not adult neural tissues, and is prominent in proliferating cells destined to be neurons and glia. It might be expected that the ~" receptor would be localized with OGF-containing pro-
liferative cells. Fifth, the ~" receptor has been identified in neural tumor cells in tissue culture 59, indicating the relationship of proliferating neural cells to this receptor. Studies now in progress using receptor ligand autoradiography and antibodies to the ~" receptor should be capable of elucidating the location of the sr receptor and complement the receptor binding data in this report. An intriguing observation recorded in the present investigation was the marked decrease in the number of ~" receptors in the cerebellum between postnatal days 10 and 11. Within a 24-h period, a 5.4-fold decrease in receptors was noted. However, the decrease in the number of layers of EGL cells in the cerebellum of .animals of postnatal days 10-12 was only about 25%. The reason(s) for the notable decrease in ~" receptors on day 11, despite a relatively small change in the number of cells comprising the EGL, is unclear. Altman ~, examining the changes in the depth of the EGL of the pyram!s in the developing cerebellum of the rat, noted that "From the eleventh day onward there is a sharp decline in the depth of the proliferative zone .... " (p. 357). If ~" receptors play a role in governing cell proliferation, it would be understandable that a decrease in the number of dividing cells would correlate with a reduction in the number of these receptors. Earlier investigations have shown that opioid receptor blockade has a profound effect on the developing organism 11,16,61-64. It is known that opioid receptor blockade often results in an up-regulation of opioid receptors, opioid gene expression, and opioid peptides 3,8,37,47-51,55,6s, and enhances physiological response to opioids 22'41'46'55'56'6s. In the present study we show that continuous opioid receptor blockade in developing animals results in an increase in body weight, confirming earlier findings6~'62'66. This regimen of receptor blockade also produces an increase in the number of g" receptors in the whole brain and in the cerebellum. Apparently, an increase in ~" receptors is a compensation for blockade of these opioid receptors. Thus, even though the ~" receptor differs in function and subcellular distribution from other opioid receptors, this receptor shares a number of properties with other opioid receptors (e.g~ ~, 8, K) such as an up-regulatory response to opioid receptor blockade, stereospecifieity, and affinity for opioid antagonists. It is also of interest to note that opioid receptor blockade results in an increase in DNA synthesis occurring in germinal neural cells64. The present data indicate that at least in one germinative matrix, the EGL of the cerebellum, the number of layers of EGL cells is substantially increased from control levels in the ccrebel-
154
lar cortex of 6-day-old rats given a continuous opioid receptor blockade. This information is consistent with previous findings that the number of neurons (and gila) are significantly increased in the brains of animals given a continuous opioid receptor blockade throughout the preweaning period 6.1'62. Our findings with respect to the ontogeny of the ~" receptor in the developing rat brain may have considerable relevance to the human situation. [MetS]enkepha lin binding sites have been identified in homogenates of human cerebellar tissue 57, with binding observed at birth and being greatest during infancy; no specific and saturable binding of radiolabeled [Met5]enkephalin was recorded in the cerebellum of adult humans. We have postulated ~1,27,58.71 that alterations in endogenous opiold systems may be related to the etiology and pathogenesis of some clinical disorders. For example, studies have reported an association between opioids and autism 13'20'24'25'27'42, self-injurious behavior 12,14, sudden infant death syndrome 3'.3s, and cancer 59'66'6s. Given the results of the present investigations, it could be hypothesized that disturbances in the ontogeny of the ~" receptor results in dysgenesis of the nervous system. Thus, an insufficient number of ~" receptors, a reduction in the affinity of this receptor, and/or a decrease in OGF might be expected to produce an increased number of cells and/or stimulation in the development of brain circuitry. This has already been documented by experiments with continuous opioid receptor blockade in the rat II,6~-63.Depending on the degree of br receptor dysfunction, neoplasia of the nervous system, the second most common cancer in children (under 15) 2, may even occur. Indeed, a strong relationship between neural cancer and [MetS]enkephalin/~ receptor has already been established 59,6z6s,72. Furthermore, an increase in the number of ~" receptors, a greater affinity of this receptor, and/or overabundance of OGF might be expected to result in a decrease in the number of cells and/or retardation in development of dendrites and synapses. Once again, depending on the severity of receptor dysfunction, impairment in the number of cells and ultimately the circuitry of the brain could result. Research on the effects of intermittent opioid receptor blockade ~1,62,63 support this contention, presumably with an opioid antagonist provoked excess of OGF interacting with an increased number of ~" receptors during the interval when the opioid antagonist is no longer available. These clinical implications only emphasize the importance of understanding how OGF and the ~" receptor participate in governing the development of the nervous system.
Acknowledgements. This work was supported by NIH Grant NS20500. We thank Dr. Tomoki Isayama for critical evaluation of the manuscript and his assistance in preparing some of the illustrations.
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