Postnatal development of multiple opioid receptors in rat brain

Developmental Brain Research, 37 (1987) 21-41 Elsevier

21

BRD50632

Postnatal development of multiple opioid receptors in rat brain Harley I. Kornblum*, Diana E. Hurlbut and Frances M. Leslie Department of Pharmacology, Universityof California, lrvine, CA 92717 (U.S.A.) (Accepted 28 April 1987)

Key words: Quantitative autoradiography; Opiate receptor;/~-Receptor; r-Receptor; h-Receptor; Ontogeny

Recent studies have demonstrated that opioid receptors may be functional at early stages of ontogeny, and may modulate specific developmental functions. It is presently unknown, however, which particular opioid receptor subtype(s) may be involved. In the preent study, we have used selective radioligand binding conditions in combination with quantitative autoradiography to examine the ontogeny of g-, r- and ~-opioid receptors in the developing rat brain. Membrane binding data indicate that the affinities of#-, r- and 6sites for radiolabeled drugs are similar in neonatal and adult rats. g- And r-receptors are present in significant densities during early neonatal periods, while h-receptors appear much later. Autoradiographic data indicate that #- and r-receptors appear early in the development of several brain regions, including the neostriatum, olfactory tubercle and rostral midbrain, and later in other regions such as the thalamus and hypothalamus. Whereas the densities of r-binding sites remain relatively constant throughout development, there is a transient appearance and/or redistribution of#-receptors in several brain areas. &Receptors are present in low densities in the basal forebrain at birth. The level of h-receptor binding increases markedly during the third postnatal week in all brain areas examined. The early appearance of#- and r-receptors during the ontogeny of the brain suggests that these receptors, at least in part, mediate the developmental actions of exogenous and endogenous opioids.

INTRODUCTION Opioid receptors exhibit a widespread distribution throughout the brain and periphery, and have been implicated in the control of a n u m b e r of physiological functions 5. These receptors mediate the biological effects of 3 large families of endogenous opioid peptides, which are localized extensively within neurons in the brain and periphery, as well as in endocrine glands 2,7. Opioid receptors appear early in the ontogeny of the mammalian central nervous system (CNS), being first detectable within embryonic rat brain by the fourteenth day (El4) of gestation 36. Autoradiographic analyses of opioid receptor distribution have indicated a changing neuroanatomical pattern at different stages of development 36'45'65. The transient appearance of both peptides 6'26 and receptors 36A5 in certain areas of the brain and spinal cord has led to the hypothesis that endogenous opioids

may subserve functional roles in the developing animal which are distinct from those in the adult. There is an increasing body of data to suggest that these endogenous opioid systems modulate the growth and development of the C N S 37'50'71. It has also been proposed that opioid receptors are involved in the regulation of other functions which are specific to the neonate, including suckling 34 and infant-maternal bonding3t,55. Opioid receptors have been subclassified into a number of pharmacologically distinct types, including/~-, 6- and r 44. However, it is as yet not clear which of these multiple opioid receptors are functional in developing brain. Although several previous studies have examined the ontogeny of opioid receptors in rodent brain 14,16,36,65these have generally focused on the/~- and t~-receptor subtypes and have involved the use of radioligands which cross-react with more than one receptor type. Thus, a heterogeneous population

* Present address: University of Hawaii, Bekesey Laboratory of Neurobiology, 1993 East-West Rd., Honolulu, HI 96822, U.S.A. Correspondence: F.M. Leslie, Department of Pharmacology, University of California, Irvine, CA 92717, U.S.A. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

22 of binding sites may have been labeled. Whereas recent studies have used highly selective binding assay conditions to characterize the developmental appearance of each receptor subtype in whole brain homogenates57"62'64, the membrane binding methodology which was used did not permit a high degree of anatomical resolution. The ontogeny of the ~-opioid receptor has proven to be particularly difficult to study owing to a tack of availability of selective radioligands. Although behavioral studies have indicated that benzomorphan drugs, such as ethylketocyclazocine (EKC), interact with a distinct ~c-opioid receptor ~9'48, membrane binding studies have shown that such compounds lack receptor selectivity and also bind with high affinity to p- and d-receptor types 12'2°'39. Whereas in guinea pig and human CNS, [3H]EKC has been clearly shown to bind to a membrane component which has the pharmacological properties of a ~c-receptor 23'39'58, there has been some controversy as to whether x-binding sites are present within rat brain 1°,32. In the present study, we have used highly selective radioligand binding conditions, in combination with the quantitative autoradiographic technique, for a detailed analysis of the ontogeny of p-, ~c- and d-receptors in rat brain, Our data indicate a differential developmental appearance of the 3 opioid receptor types. Whereas p- and ~c-receptors are present at an early stage of postnatal ontogeny in a pattern which changes with age, d-receptors appear at later stages of development in a pattern similar to that of the adult. MATERIALSAND METHODS

Animals Adult and pregnant female rats were obtained from Simonsen (Gilroy, CA) and kept on a 12 h light-dark cycle. The day of birth was designated P0. Membrane binding Male rats of various ages (P3-60) were sacrificed by decapitation, their brains removed and homogenized in ice-cold Tris-HC1 buffer (50 raM, pH 7.4). Homogenates were centrifuged at 17,000 g for 15 min, and the resulting pellets resuspended in buffer and incubated at 37 °C for 1 h to facilitate dissocia-

tion of bound endogenous ligand. The brain membranes were then washed 3 times by centrifugation (17,(/00 g for 15 min) and resuspended in fresh buffer. Membrane homogenates (10 mg original tissue wet wt./mi) were incubated for 60 min at 22 °C in a final volume of 1 ml Tris-HCl buffer (50 mM, pH 7.4) containing radioligand and, where appropriate, competing drug(s). Selective labeling of p-, d- and ~c-binding sites was achieved as follows. For p-sites, the highly selective radioligand [3H][D-AIa2,MePhe4, Gly-olS]-enkephalin (DAGO) (45-60 Ci/mmol; Amersham, IL) was used. For d-sites, [3H][D-Ala2,DLeuS]-enkephalin (DADLE) (29-54 Ci/mmol; Amersham, IL), which binds to both p- and d-receptors 42, was used in combination with sufficient [DPro4]-morphiceptin (300 nM) to totally suppress/~receptor binding. In some experiments, the total population of ~- and d-receptors were labeled using [3H][D-Ala2]-Met-enkephalinamide (DAMA) (2050 Ci/mmol; Amersham, IL). For selective labeling of ~c-sites, [3H]EKC (18-25 Ci/mmol; New England Nuclear, MA) which binds to all 3 receptor types 2°'39, was used in combination with sufficient unlabeled [D-Pro4]-morphiceptin (300 nM) and [D-Set 2, Thr~']Leu-enkephalin (DSLET, 10(1 nM) to completely suppress binding to p- and &receptors, respectively. Following incubation, membrane bound radioligand was separated by filtration through Whatman GF/C filters, which were then rinsed with 3 x 5 ml aliquots of ice-cold buffer. Retained radioactivity was measured by liquid scintillation spectroscopy. Specific binding was defined as the difference in binding in the absence and presence of levallorphan (1 pM). Radioligand binding constants (K,t and Bmax) were determined by analysis of saturation curves using the non-linear, least squares regression LIGAND program 54. Equilibrium dissociation constants (K~ values) for a number of unlabeled, competing ligands were determined by analysis of dose-response curves for inhibition of radioligand binding, using the statistical, non-linear curve fitting procedure of Richardson and Humrich 6~. The inhibitors which were used in the study included DAGO, DADLE, morphiceptin, fl-endorphin, dynorphin A (all obtained from Peninsula Labs, Palo Alto, CA), and U50488H (Upjohn, Kalamazoo, MI). [D-Pro4]-Morphiceptin and DSLET were also obtained from Peninsula Labs.

23

Receptor autoradiography A modification of the technique of Herkenham and Pert 29 was used to determine the autoradiographic distributions of opiate receptors in developing and adult brains. Brains from male and female animals of ages P0 to P60 (n = 2-3 per group) were removed and frozen in isopentane for 30 s at -30 °C. Twenty-micrometer sections were cut at -20 °C and thaw-mounted onto subbed glass slides. The sections were dried for 2 h at 0 °C, and stored at -20 °C until use.

In preliminary experiments, in which tissue-bound radioactivity was quantitated by liquid scintillation spectroscopy, incubation and washout parameters were determined empirically such that the ratio of specific to non-specific binding was optimized while maintaining equilibrium binding. Using the incubation conditions outlined below, specific binding, as determined in the absence and presence of levallorphan (1 pM), was 90-98% for [3H]DAGO and [3H]DADLE, and 75-90% for [3H]EKC. Radioligand K s values and pharmacological displacement profiles were similar to those determined in membrane homogenates (data not shown). For autoradiographic visualization of radioligand binding sites, incubations were carried out simultaneously for all sections being compared directly. Slides were preincubated for 15 min at 22 °C in Tris buffer (50 mM, pH 7.4) containing 100 mM NaC1, 2 pM GTP and 1% BSA. The sections were then rinsed for 30 s in 50 mM Tris buffer. Following preincubation, p- or 6-binding sites were selectively labeled by incubation with [3H]DAGO (1.6 nM) or [3H]D A D L E (2.0 nM) plus [D-Pro4]-morphiceptin (300 nM), respectively. Following a 60-min incubation at 22 °C, the tissue sections were rinsed in two changes of ice-cold buffer for 10 min, and rapidly dried under a stream of cool air. K-Binding sites were selectively labeled by incubation for 90 min at 22 °C in 50 mM Tris buffer (pH 7.4) containing [3H]EKC (2.0 nM), with unlabeled [D-Pro4]-morphiceptin (300 nM) and DSLET (100 nM) to suppress binding to p- and 6-receptors, respectively. Tissue sections were then rinsed in 3 changes of ice-cold buffer for 15 min, and rapidly dried under a stream of cold air. For all radioligands, alternate sections were incubated in the absence and presence of levallorphan (1 pM) to define non-specific binding.

Following incubation and air-drying, the slides were desiccated overnight, then mounted with radioactive brain paste standards (prepared with [3H]thymidine, ICN), in close apposition to a sheet of tritium-sensitive film (Ultrofilm [3H], LKB). After an exposure period of 6-10 weeks for p- and 6-sites, or 12-24 weeks for K-sites, the latent autoradiographic image on the film was developed and tissue sections were stained with Cresyl violet. In all brains, receptor distributions were determined qualitatively by comparison of autoradiograms with the Nissl-stained sections. In a series of brains from animals aged P2-P43, binding densities were quantitated using computer-assisted video-image analysis as described by Altar et al. 3. Using the radioactive brain paste standards a calibration curve relating image intensity to the amount of radioactivity was generated. This standard curve was then used for linearization and quantitation of autoradiographic images. Specific binding values were determined for individual regions under the manual control of the operator. Data represent the averaged mean of 5-20 measurements per brain area and are expressed as fmol radioligand bound/mg protein. RESULTS

Membrane binding studies p- And 6-sites. K a and Bmax values for the binding of [3H]DAGO, [3H]DADLE (with p-binding suppressed) and [3H]DAMA to membrane homogenates of neonatal (P6) and adult rat brain were determined by computer-assisted LIGAND analysis of saturation curves (Table I). The best statistical fit of all data was to a single site binding model. There were no significant differences in radioligand K d values in neonatal and adult brain tissue. In contrast, Bmax values for all radioligands were significantly higher in adults. The concentration of 6-opioid binding sites, as labeled with [3H]DADLE in the presence of [o-Pro4]-morphiceptin, was very low at P6, and exhibited a 10-fold increase in adults, p-Binding sites, as labeled with the p-selective radioligand, [3H]DAGO, were present in significant number at P6, and exhibited a smaller, 3.5-fold increase in adults. At both P6 and in the adult, the maximal binding capacity for [3H]DAMA was, within experimental error, equivalent to the sum of that of [3H]DAGO

24 TABLE 1

Radioligand binding constants in neonatal and adult rat brain membranes Brain membranes from neonatal and adult rats were incubated with 8-10 concentrations of radioligand as described in the text. At each radioligand concentration, specific binding values were calculated as the difference in bound radioactivity in the absence and presence of levallorphan ( 1/tM). Equilibrium dissociation constants (Kd) and binding site densities (Bmax) were determined using the non-linear regression program, LIGANDS! All values represent means _+ S.E.M. of (n) determinations.

P6 Kd (nM) Brnax(pmol/g wet wt. ) Adult Kd(nM ) Bmax (pmol/g wet wt.)

[3H]DAGO

[3H]DADLE

[3H]DAMA

[JH]EKC

0.70 _+0.22 (3) 3.1 + 0.6*

2.47 + 0.79 (3) 0.5 ___0.1"

0.84 + 0.18 (4) 3.5 + 0.7*

0.97 + 0.48 (3) 0.94 + 0.34

0.68+0.06(3) 10.8 ± 0.7

2.11+0.45(5) 5.9 + 0.7

1.11+0.19(7) 15.0 + 1.4

0.92+0.32(4) 1.80 + 0.60

* Denotes statistically significant differences between values in P6 and adult, P < 0.01, Student's t-test.

a n d [ 3 H ] D A D L E (see T a b l e I). T h e s e data are consistent with those of p r e v i o u s studies 2t'44, which have indicated that carboxyl t e r m i n a l a m i d a t e d analogs of e n k e p h a l i n , such as D A M A , b i n d with almost e q u a l affinity to/x- a n d cLsites. This lack of r e c e p t o r selectivity was f u r t h e r c o n f i r m e d by i n h i b i t i o n studies, in which the K i values for D A M A d i s p l a c e m e n t of [3H]DAGO and [3H]DADLE binding (with/x-binding suppressed) were f o u n d to be 1.3 + 0.1 a n d 1.9 + 0.1 n M (n = 3), respectively. W h e r e a s [ 3 H ] D A M A b i n d i n g was c o m p l e t e l y inhibited by D A D L E , a p e p t i d e with high affinity for both/x- a n d 6-sites (data n o t s h o w n ) , it was i n c o m p l e -

tely displaced by the/x-selective agonist, m o r p h i c e p tin (Fig. 1). T h e m a x i m a l i n h i b i t i o n achieved by m o r p h i c e p t i n d e c l i n e d with age, indicating an i n c r e a s e in the n u m b e r of 6-receptors as a p e r c e n t a g e of total [ 3 H ] D A M A b i n d i n g . A highly significant increase in the p e r c e n t a g e of 6-receptors o c c u r r e d b e t w e e n P12 a n d P15 ( T a b l e II). ~c-Sites. A f t e r s u p p r e s s i o n of b i n d i n g to/x- a n d 6sites with selective c o m p e t i n g ligands, a small resi-

TABLE II

Relative proportion of kt and D-binding sites at different developmental stages Brain membranes from rats of various ages were incubated with [3H]DAMA (1 nM) in the presence of 8-10 concentrations of the/~-selective agonist, morphiceptin. Dose-response curves for displacement of [3H]DAMA binding were constructed and analyzed by the non-linear regression program of Richardson and Humrich61. Values are means + S.E.M. of (n) observations.

~x

|-

Morphlceptin concemrmlon (M)

Fig. 1. Dose-response curves for displacement of specific [3H]DAMA binding by the/~-selective agonist, morphiceptin, at various stages of postnatal development. Brain membranes from rats ages P6 (closed squares), P15 (closed circles) and P25 (open squares) were incubated with [3H]DAMA in the presence of various concentrations of morphiceptin. The dose-response curves which are illustrated are representative examples of triplicate experiments.

Age ( d a y s )

D-Binding(% total l~ + ~)a

6:l~-Ratio

3 6 9 12 15 18 21 25 Adult

5.7 _+0.3 (3) 8.7 + 1.5 (3) 8.0 + 2.1 (3) 13.0 + 1.5 (3) 21.7 + 0.9 (3)* 22.0 + 3.6 (3) 25.7 + 2.8 (3) 26.2 + 2.4 (4) 32.5 + 2.2 (6)

1:16.5 1:10.5 1:11.5 1:6.7 1:3.6 1:3.5 1:2.9 1:2.8 1:2.1

a Represents the morphiceptin non-displaceable component as a percentage of total specific [3H]DAMA binding. * Denotes a statistically significant difference from the previous age group, P < 0.01, Student's t-test.

25 dual component of [3H]EKC binding remained. Computer analysis of saturation curves indicated that the statistical fit of the data to a one site model was equal to, or better than, that of a two site model. The more conservative one site model was therefore preferred. K d values for radioligand binding to this site were not significantly different in neonatal and adult rat brain (Table I). Although the total number of binding sites increased by a factor of two from P6 to adulthood, this difference was not found to be statistically significant (P > 0.05, Student's t-test). The pharmacological profile of the non-p-, non-6component of [3H]EKC binding in adult rat brain was found to be consistent with that of a x-opioid receptor. Drugs which have low affinity for x-binding sites, such as D A G O and D A D L E 22'39, produced little or no displacement of radioligand binding (Table III). In contrast, drugs which have been reported to have high affinity and/or selectivity for x-receptors, such as dynorphin A and U50488H t2'41, were potent inhibitors of binding. The rank order of potencies for displacement of [3H]EKC binding was dynorphin A > U50488 H > fl-endorphin > > > D A G O = DADLE (Table III). A utoradiographic studies Adult distribution. The autoradiographic distribu-

tions ofp-, 6- and r-binding sites in adult rat brain are

TABLE III Inhibition constantsfor displacement of the non-t~-, non-O-component of [3H]EKC binding

Brain membranes from adult rats were incubated with [3H]EKC (2 nM) in the presence of [D-Pro4]-morphiceptin(300 nM) and DSLET (100 nM), and 8-10 concentrations of each inhibitor as described in the text. Dose-response curves for displacement of the non-/~-, non-0-component of [3H]EKC binding were constructed and analyzed by the non-linear regression program of Richardson and Humrich61. Values represent means + S.E.M. of (n) observations. Displacer

Ki (nM)

Dynorphin A U50488H fl-endorphin DAGO a DADLEa

0.58 + 0.19 (4) 25.7 + 9.5 (4) 30.6 + 1.8 (4) >1000 (3) >1000 (3)

a

Denotes that DAGO and DADLE, at concentrations of 1 0 - 6 M, inhibited radioligand binding by 19.2 + 10.2 and 23.3 + 4.7%, respectively.

illustrated in Fig. 2. The anatomical localization of pand 6-receptors, as determined in the present study, is very similar to that described previously28'29'46'47' 49,66. As the following description indicates, the autoradiographic distribution of r-binding sites is similar, but not identical, to that of p-receptors. Telencephalon. While there are significant densities of p- and 6-receptors in olfactory bulb, there is little labeling of x-binding sites in this region (Fig. 2A-C). p- And 6-binding sites exhibit a differential pattern of distribution, p-Receptor binding is distributed in the glomerular, external plexiform (EPL) and internal plexiform layers (IPL) of the main olfactory bulb, and in much higher density in the accessory olfactory bulb (AOB) (Fig. 2A, arrow). There is a dense region of binding in the pars externa of the anterior olfactory nucleus (Fig. 2A, arrowhead). Delta receptor binding, is concentrated within the EPL, with low-moderate amounts within the granule cell layers, IPL and the anterior olfactory nucleus (Fig. 2C). All 3 opioid receptor types are labeled in the olfactory tubercle (Fig. 2D-F). p-Receptor binding is largely confined to small 'patchy' areas just ventral to the nucleus accumbens, with a much lower 'background' level of binding (Fig. 2D). Such p-opioid receptor patches have been described previously28, and appear to be of striatal origin. Moderately high levels of x- and 6-receptor binding also occur within the olfactory tubercle, with little or no patchiness detectable (Fig. 2E,F). There is significant labeling of all 3 opioid receptor types within the adult striatum (Fig. 2D-F). There is a moderately dense area of p-receptor binding in the medial nucleus accumbens, with a more dense area associated with the major island of Calleja (insula magna) at the medial border of this structure (Fig. 2D, arrow). The lateral nucleus accumbens and the caudate-putamen exhibit the classic patchy pattern of binding described by others 56. The distribution of xreceptor binding in this region resembles that of p-. There is a relatively high density of r-receptor binding in the medial nucleus accumbens, with lower levels in the portion lateral to the anterior commissure (Fig. 2E). At more rostral levels of the caudate-putamen, 'patches' of dense binding are apparent on a background of less than 50% of that binding density. There are higher levels of x-receptor binding in some

27 portions of the subventricular zone, just lateral to the lateral ventricle (not shown). &Receptor binding in the nucleus accumbens and caudate-putamen is nonpatchy (Fig. 2F). While there are high levels of 6-receptor binding in the medial nucleus accumbens and lateral caudate-putamen, binding levels in the lateral nucleus accumbens and medial caudate-putamen are somewhat lower. There is relatively little labeling of /t-, r- or d-receptors in the globus pallidus and entopeduncular nucleus. While densities of all 3 opioid receptor types are generally low throughout the septum, there are regions of elevated/t-receptor density in the medial septum and dorsolateral septum (Fig. 2D). There is significant labeling of/t-, r- and &binding sites in the bed nucleus of the stria terminalis. p-Receptor binding in the neocortex is as described by Lewis et al. 43, with superficial, intermediate and deep layers generally corresponding to layers I; III, IV or V (depending on brain region); and VI, respectively (Fig. 2D). r-Receptor binding in the neocortex is generally light (Fig. 2E). There are slightly higher levels of binding in two bands, a deeper band, corresponding to layers V and VI, and a more superficial one, corresponding to layer III or IV. Some binding also appears to be associated with layer I. There is, however, a more dense layer of binding in the deep portion of the gustatory area of the neocortex. There are also relatively high levels of r-receptor binding in the claustrum (Fig. 2E, arrow) and endopiriform nucleus, d-Receptor binding in the neocortex is non-uniform (Fig. 2F). There are two broad bands of binding, internal and external, separated by a thinner band of much lower binding density. There is also a limited amount of binding in the deepest cortical layer. The limitations of the film technique have, however, prevented the precise determination of cortical layers. There are moderately high levels of &receptor binding in the claustmm, but not the endopiriform nucleus.

There are similar distributions of/t- and r-binding sites across the amygdaloid nuclei. Highest levels of binding are in the posterior cortical and medial nuclei, and in the intercalated masses, with lower levels in the basolateral and lateral nuclei. However, the relative density of/t-receptor binding in the basolateral nucleus (Fig. 2G, arrow) is much greater than that of r-receptors. &Receptor binding in the amygdala is largely confined to the basolateral, posterior cortical and medial nuclear groups. The hippocampal formation contains high densities of p-binding sites, with significantly lower densities of 6- and r-sites. Highest levels of p-receptors are localized in the CA 3 region of the stratum pyramidale (Fig. 2G, arrowhead), with lower levels in the CA 1 region, stratum lacunosum moleculare and the granule cell layer of the dentate gyms. &Binding sites are present in some areas of the hippocampus and dentate gyrus; however, there is less heterogeneity of binding density between regions (Fig. 2I). The density of r-receptors in the dentate gyrus and most of the hippocampus is quite low (Fig. 2H). However, the pattern of binding is similar to that of/t-receptors, with a higher density in the CA 3 pyramidal cell region than in CA t. There is a higher level of r-receptor binding in the stratum lacunosum moleculare than in the pyramidal cell layer (Fig. 2H). There are relatively high levels of both/t- and r-binding in the presubiculum. Diencephalon. While there are very low levels of d-receptor binding throughout the thalamus, hypothalamus and midbrain, these areas contain significant densities of/t- and r-sites (Fig. 2G-I). With the exception of certain regions such as the parafascicular and ventroposterior nuclei, the dorsal thalamus is densely labeled by p-binding sites. High levels of binding occur in several nuclei, including ventrolateral, reticular, lateral dorsal, posterior, and dorsoand ventrolateral geniculate with lower levels in the medial geniculate nuclei. Slightly higher levels occur

Fig. 2. Distribution of p- (A,D,G,J), ~c-(B,E,H,K) and d (C,F,I,L)-opioid binding sites in some regions of adult rat brain. Sections were incubated with radioligand as described in Materials and Methods, and apposed to tritium-sensitivefilm. The images presented here are printed directly from the resulting autoradiograms. Arrow in A points to the accessoryolfactory bulb, while the arrowhead points to the external anterior olfactory nucleus. Arrow in D points to the major island of Caileja (insula magna) at the medial border of the nucleus accumbens. Arrow in E points to the claustrum. Arrowhead in G points to the CA3layer of the hippocampus, while the arrow points to the basolateral amygdala. Arrow in H points to the zona incerta. Arrow in J points to the superficialgray layer of the superior colliculus.

28 in the central medial, mediodorsal and midline nuclei. There is little binding in the zona incerta. There is a low density of ~¢-binding sites in much of the dorsal thalamus, with the exception of the central medial, mediodorsal and midline nuclei. There are low to moderate levels of binding in the posterior nucleus, the lateral posterior nucleus, the dorsolateral and medial geniculate nucleus, with very little binding in the ventral posterior nuclei. There is a moderate density of r-binding sites in the thalamic reticular nucleus and zona incerta (Fig. 2H, arrow). This pattern of binding appears to be continuous with that of lateral hypothalamus. There are high densities of both p-and K-binding sites in the medial habenula and within the subfornical organ of the circumventricular system. Relative to the thalamus, the density of r-binding sites is higher in the hypothalamus while that of pbinding sites is lower. /~-Receptor binding is fairly sparse throughout much of the hypothalamus, with the exception of the anterior hypothalamic area, the suprachiasmatic, dorsomedial and ventromedial nuclei, and the supramammillary region. In contrast, there is a relatively high density of r-receptor binding throughout the hypothalamus with the exception of the mammillary bodies. The densest regions of binding include the medial preoptic area, and the paraventricular, supraoptic and suprachiasmatic nuclei. Mesencephalon. While there is very sparse labeling of b-binding sites within the mesencephalon, there are significant densities of both p- and 7c-binding sites in a similar pattern of distribution (Fig. 2J-L). The superficial, intermediate and deep gray layers of the superior colliculus contain both p- and ~cbinding sites. The relative level of receptor binding is higher within the superficial gray layer (Fig. 2J, arrow). There are moderate levels of both p- and ~:binding sites in the external nucleus of the inferior colliculus, and in the periaqueductal gray. The interpeduncular nucleus represents one of the densest areas of both p- and x-binding in the entire brain. In the substantia nigra, there are moderately high densities of p-binding sites in the pars compacta and the external portion of the pars reticulata, with lower levels of ~c-binding sites exhibiting a more uniform pattern of distribution.

Ontogeny Autoradiograms and quantitative analyses of opi-

oid receptor development in various brain regions are illustrated in Figs. 3-9. Although pups of both gender were used for analysis of the ontogeny of pand b-sites, there were no quantitative differences between male and female in any of the brain regions examined. Telencephalon. The ontogeny of p- and b-receptors in the olfactory bulb is shown in Fig. 3. At birth, p-receptor binding is very high in the EPL and AOB. Binding in the EPL declines markedly between P2 and P9. In the AOB, binding remains at moderately high levels throughout development, but does drop by 50% between P5 and P9. Binding in the glomerular layer is detectable by P13 and increases to adult levels by P17. By P5, #-receptor binding is detectable in the IPL and the anterior olfactory nucleus, and remains relatively constant throughout postnatal development, b-Receptor binding is almost completely absent at birth in the olfactory bulb. Binding appears within the EPL between P9 and P13, and increases in density throughout development. Binding in the granule cell layer, although much lower in density, follows a similar time course. There is litte 7c-receptor binding within the olfactory bulb at any stage of development. p-, ~c- And b-binding sites are all detectable in the olfactory tubercle at birth, although p-receptor density is much higher than that of the other opioid receptor types (Figs. 4 and 5). p-Receptor binding markedly decreases in density between P2 and P13, with the exception of binding in patches just ventral to the nucleus accumbens (Figs. 4A,C,E and 5A). The density of ~c-binding sites within the olfactory tubercle remains constant throughout development (Figs. 4B,E,H,K, and 5B), while that of b-binding sites increases, with a 3-fold rise occurring between P9 and P13 (Figs. 4C,F,I and 5C). #-Receptor binding is densely localized in the medial nucleus accumbens at birth (Fig. 4A, arrowhead, and Fig. 5D). This binding diminishes approximately 4-fold in the first month of life, but remains at moderately high levels. K-Receptor binding is present within this region at birth, remaining at constant density throughout development (Figs. 4B,E,H,K and 5E). Low levels of b-receptor binding sites are also visible within the medial nucleus accumbens at P2, increasing to almost adult levels within the following two weeks (Figs. 4C,F,I,L and 5F).

29

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Fig. 3. Ontogeny of p- (A,C,E,G) and 6 (B,D,F,H)-binding sites in the olfactory bulb. Sections from animals of the indicated ages were prepared for autoradiography as described in Materials and Methods. Autoradiograms (A-F) are photographs of the computerprocessed, linearized images used for quantitation. A: arrow, EPL. C: arrowhead, IPL. D: arrow, EPL. E: arrowhead, glomerular layer (GL). F: arrowhead, granule cell layer. G, H: represent quantitative analysis of developmental changes in the densities of#- and 6-binding sites, respectively.

30

Fig. 4. Ontogeny of p- (A,D,G,J,) ~¢-(B,E,H,K) and 6- (C,F,I,L) binding sites in the anterior forebrain. Sections from animals of the indicated ages were prepared for autoradiography as described in Materials and Methods. The autoradiographic images are photographs of the computer-processed, linearized images used for quantitation. Note that the degree of enhancement is the same for all sections incubated with one ligand, but is different for sections incubated with different ligands. A: arrow, medial nucleus accumbens; arrowhead, olfactory tubercle. B: arrow, ventrolateral striatum.

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Fig. 5. Quantitative analysis of the ontogeny of/~- (A,D,G), •- (B,E,H) and 6- (C,F,I) binding sites in anterior forebrain. Sections from animals of the indicated ages were prepared for autoradiography as described in Materials and Methods. At each age, specific binding of radioligand in each of the indicated brain regions was determined by computer-assisted quantitative analysis of the autoradiographic images. A-C: olfactory tubercle. D-F: medial nucleus accumbens. G-I: anterior caudate-putamen. Closed squares, dorsal matrix; open squares, dorsal patches; closed triangles, ventrolateral streak (VL); closed diamonds, lateral matrix; open diamonds, medial matrix.

Prior to birth,/~-receptor binding within the caudate-putamen is extremely dense and appears in a homogenous distribution, as described by Kent et al. 36. By the time of birth, binding within this region has become somewhat patchy, although binding within the background matrix between patches is still moderately high (Figs. 4A and 5G). Between P2 and P13, binding levels in the patches increase two-fold,

while that within the matrix drops to almost undetectable levels. At birth, there is a dense streak of ~¢-receptor binding along the ventrolateral striatum (Fig. 4B, arrow, and Fig. 5H), which is visible throughout development in the same density as in the neonate, although the pattern becomes more circumscribed. There is also a homogeneous distribution of 'matrix' or non-patchy binding throughout the anterior cau-

32 date-putamen, which remains at relatively constant density during the first month of postnatal development. In contrast, binding in striatal patches and in the subependymal region of the caudate-putamen appears during the third postnatal week (Fig. 5G). 6Receptor binding in the striatum does not appear to be patchy at any time during development. There is very little binding present in this region at birth, with a marked increase occurring between P13 and P17. A lateral to medial binding gradient is present through-

out development, with highest levels occurring in the lateral caudate-putamen (Figs. 4C,F,I,L and 51). /,-Receptor binding is present in the medial septum and nucleus of the diagonal band at birth, and detectable in the lateral septum soon thereafter. This binding remains constant during development, with little change in density following an increase in the first week of life. /~-Receptor binding in the globus pallidus and entopeduncular nucleus changes markedly during de-

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33

(Fig. 7B). Binding in the endopiriform nucleus appears slightly earlier (P6) than that in the daustrum nucleus (P9), although both reach maximal densities by P17 (Fig. 7B). b-Receptors also appear in a nonuniform manner within the neocortex (Fig. 7C). Binding initially appears within deep cortex during the first postnatal week. Binding in more superficial regions is detectable by P9. Between P13 and P17, marked increases occur in both the deeper and more superficial layers. /~-Receptors are present in high densities in the amygdaloid nuclei at birth. The levels of binding then increase over the next 3 weeks to those seen in the adult (Fig. 7D). r-Binding in the amygdala appears differentially across nuclei (Fig. 7E). Whereas binding is apparent in the basolateral complex at P5, it is not detectable within other nuclei until P12 or P17. bReceptors in the amygdala are detectable in low densities at birth. Binding gradually increases in intensity over the following postnatal weeks (Fig. 7F). The ontogeny of ~t-receptor binding in the hippo-

velopment, as shown for the globus pallidus in Fig. 6. Binding in these areas is very high during the first week of life, and undergoes a 25-fold decrease following the second postnatal week. Moderately dense r-receptor binding appears within both pallidal nuclei during the second postnatal week, and falls by approximately 50% between P12 and P17 (data not shown). In contrast, b-receptor binding first becomes apparent at low levels within the globus pallidus by P9, and decreases only slightly during development. In the neocortex, there is a differential appearance of p-receptor binding in different laminae as described by Kent et al. 36. While binding is apparent in layers I and VI during the first postnatal week, that in the middle layer (layer III, IV, or V, depending on the brain region 43) does not appear until after P9 (Fig. 7A). There is a marked increase in binding within all layers between P13 and P17. r-Binding in most of the neocortex appears relatively late, with significant levels reached only by P12. Binding in the deep gustatory cortex, however, is apparent by P9

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campal formation is in agreement with that found by Unnerstall et al. 65. At birth, #-receptors are diffusely distributed in low density throughout the hippocampus. By the end of the first week, binding is apparent in discrete laminae corresponding to the stratum pyramidale and the stratum lacunosum moleculare. During the second week, binding in stratum pyramidale increases to near-adult levels, r- And b-binding sites, only faintly visible in the adult, are detectable in the hippocampus by the end of the second postnatal week in extremely low densities (data not shown). Diencephalon. In most regions of the hypothalamus, p-receptor binding is present in low density at P2, with the exception of binding in the dorsal hypo-

thalamus, which exhibits quite high receptor densities. Binding throughout much of the hypothalamus increases during the first two weeks, and then falls off to lower adult levels during the following two weeks (Fig. 8A). Binding in some areas, such as the anterior hypothalamus, remains at moderate levels throughout development, r-Binding sites first appear in the hypothalamus at P6, reaching adult densities in the supraoptic and paraventricular nuclei by P12, and in most other region by P17 (Fig. 8B). In the thalamus, p-receptors appear quite late during development, with the exception of the ventral thalamus and midline nuclei. Binding in the dorsal thalamic nuclei reaches appreciable levels by P13

35

with the characteristic pattern of laminations becoming apparent by P17./z-Receptor binding is present in both the central gray and the surrounding tegmental region at birth, increasing in intensity until P13. The density of/z-binding sites in both regions then decreases, approaching undetectable levels within the tegmentum by P43 (Fig. 9C). In contrast, r-receptor binding within the central gray increases during the first 3 postnatal weeks, then remains at constant levels, while binding within the tegmentum peaks at P12 and then decreases slightly (Fig. 9D). There is no significant labeling of fl-binding sites within the mesencephalon at any stage of development.

(Fig. 8C), and increases to almost adult levels by P17. Binding in the medial habenula is very dense at birth and remains so into adulthood, r-Receptor binding is first detectable within the ventral and midline thalamus by P6, and in the rest of dorsal thalamus by P12 (Fig. 8D). Binding densities in these regions slowly increase to adult levels by P38. Mesencephalon. Both/z- and r-receptor binding are detectable within the substantia nigra, pars reticulata, during early postnatal development (Fig. 9A,B). Whereas the density of r-binding sites remains constant, there is a two-fold decline in/z-receptor binding within this region following the fourth postnatal week. Both/Z- and r-binding sites are apparent within the superior colliculus during the first postnatal week, in a homogeneous pattern of distribution. The densities of both types of receptor increase during the following two weeks (Fig. 9A,B),

DISCUSSION Using pharmacologically selective radioligand binding conditions, we have examined the ontogeny

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36 of p-, ~c- and b-opioid receptors in rat brain. LIGAND analysis of saturation binding data has indicated that p-sites, labeled by [3H]DAGO, and bsites, labeled by [3H]DADLE in the presence of [DPro4]-morphiceptin, increase in number during development, but do not change significantly in affinity. In this latter finding, we are in disagreement with Spain et al. 62 who have found that d-receptors have a much lower affinity for [3H]DADLE in neonatal rats than in adult. In contrast, our data indicate that there are very low numbers of d-binding sites in the brains of neonatal rats, but that these are of similar affinity to those of the adult. The reason for this discrepancy is unclear, although it may reflect differences in experimental methodologies. As has been emphasized by Molinoff et al. 5~'5:, one useful approach for quantitation of heterogeneous binding sites is the use of non-selective radioligands in combination with highly selective competing ligands. In the present study, we have applied this technique for more detailed analysis of the developmental appearance of p- and d-binding sites in tissue homogenates. [3H]DAMA fulfills the critrion as a non-selective radioligand, in that it has almost equivalent affinities for p- and d-binding sites. Thus, it labels the total population of p- and d-receptors. The fraction of d-receptors, as a percentage of total [3H]DAMA binding, may then be calculated by analysis of dose-response curves for inhibition of radioligand binding by p-selective agonists, such as morphiceptin. Using this approach, we have characterized a gradual increase in the ratio of &p-binding sites throughout postnatal development, with a particularly significant increase occurring between P12 and P15. These findings are consistent with the results of our autoradiographic study. The present study also provides evidence that [3H]EKC binds, with high affinity, to an opioid site within rat brain which has properties distinct from that ofj~- or b-receptors. Although it is present in low concentrations in adult brain tissue, this site is consistently detectable when radioligand binding to p- and &receptors is suppressed by co-incubation with selective competing ligands. These data contrast with those of Hiller and Simon 32, who were unable to detect a non-p-, non-d-component of [3H]EKC binding in rat brain. They are, however, consistent with the findings of others 20~46"4753"57-59"62"63"69. Our present

data indicate that this [3H]EKC binding site has pharmacological properties which are consistent with that of a ~c-opioid receptor. Radioligand binding is potently displaced by dynorphin A, which has been shown to bind with high affinity to the ~¢-opioid receptor j2. It is also inhibited by low concentrations of U50488H, a synthetic agonist which exhibits a high degree of ~¢-receptor selectivity41. While it has previously been suggested that the non-p-, non-b-component of binding of benzomorphan drugs (such as EKC) in rat brain tissue represents a/3-endorphin-selective receptor 1°, our present data do not support this hypothesis. We have found that the potency of/~-endorphin to inhibit [3H]EKC binding is more than one order of magnitude lower than that of dynorphin A, an opioid peptide which has not previously been reported to have high affinity for the e-receptor subtype. Furthermore, we have found that fl-endorphin inhibits [3H]EKC binding to rat brain in a concentration range which is similar to that previously reported for inhibition of h:-receptor binding in guinea pig brain 6°. Using the same, selective.radioligand binding conditions, we have characterized the autoradiographic distributions of p-, ~c- and b-receptor binding in the developing and adult rat brain. Since single concentrations of radioligand were used for this analysis, the observed ontogenetic changes in autoradiographic grain densities may reflect changes in either the number (Bmax) or the equilibrium dissociation constant (Kd) of the binding sites. Given our findings, and those of others 57'64, that receptor Ka values in membrane homogenates do not change significantly throughout ontogeny, we presume that the changes in autoradiographic grain density observed during development represent changes in receptor number. A much more detailed pharmacological analysis would be required, however, in order to confirm this assumption. In adult rat brain, the distribution of/~- and d-receptors which we have observed is largely similar to that described by others 28'29'46"47'49'65'66.The distribution of d-receptors in adult brain is very different from that of p-receptors. The relatively homogeneous distribution in the neostriatum, nucleus accumbens and olfactory tubercle is in stark contrast to the patchy distribution of p-receptors, d-Receptor binding is also almost entirely absent in the diencephalon and brainstem, with the exception of a very

37 dense area of binding in the pontine tegmentum. This pattern of p- and d-receptor labeling is more circumscribed than that observed in early studies in which less selective binding conditions w e r e u s e d 24'43. Our data correlate extremely well, however, with that of more recent studies in which stringent precautions were taken to limit cross-reactivity of radioligand binding46,47,49. Whereas previous reports have indicated that the distributions of p- and x-receptors in adult rat brain are indistinguishable 59'6° our present findings indicate that there are significant differences in their patterns of anatomical localization. Areas in which the pattern of x-receptor distribution is similar to that of p-receptors include neostriatal 'patches', medial nucleus accumbens and mesencephalon. In contrast, there are marked differences in the distribution of pand r-binding sites in the neocortex, olfactory tubercle, thalamus and hypothalamus. This distribution of x-receptors is generally consistent with the recent reports of others 46,47,53, in which [3H]bremazocine was used as radioligand. One interesting difference, however, is our observation of x-receptor patches within the caudate-putamen. While it is possible that these patches are the result of incomplete suppression of [3H]EKC binding to p-receptors 47, we consider this unlikely as the pattern of x-receptor development within striatal patches differs from that of p- (see below). Since several recent reports have postulated the existence of r-receptor subtypes 4,9,25,58, it is possible that[3H]EKC labels an additional x-receptor population which was not labeled by [3H]bremazocine under the assay conditions used in other studies. Our membrane binding data indicate that both pand x-binding sites are present in significant concentrations in neonatal rat brain, in agreement with the findings of others 57,62. However, the ontogeny of xreceptors differs from that of p-. While there is a highly significant increase in p-receptor density in adult rat brain, there is a much smaller increase in xreceptor density. Such findings are consistent with those of our autoradiographic studies, in which marked differences in the developmental appearance of p- and x-receptors are seen. In the forebrain of the newborn rat, x-receptor binding is present in high densities as a streak along the ventrolateral striatum, and within the olfactory tubercle and medial accumbens. p-Receptors at this age, however, are present

in high density throughout the striatum, in addition to the olfactory tubercle and accumbens, p-Receptor patches appear during the first postnatal week, with a concomitant decrease of 'matrix' binding in the neostriatum and olfactory tubercle, whereas r-receptor patches are not visible until the third week. There is no loss of 'matrix' binding within the caudate-putamen while the r-receptor patches are forming. In other regions, such as the EPL or the olfactory bulb, medial nucleus accumbens, 'non-patch' neostriatum and olfactory tubercle, and hypothalamus, there is a transient developmental appearance of p-binding sites, while the densities of r-binding sites remain constant or increase to adult levels. In some brain regions, however, there are similarities in the developmental appearance of p- and xbinding sites. In the mesencephalon, both p- and xbinding sites are present at birth and generally follow a similar developmental time course. In the thalamus, both p- and x-binding sites appear toward the end of the first postnatal week, and increase to adult densities during the following two weeks. In certain areas, such as the globus pallidus, entopeduncular nucleus and midbrain tegmentum, both p- and xbinding sites are present in moderately high densities at early stages of postnatal development, then decrease to lower adult densities during the first postnatal month. The ontogeny of d-receptor binding is very different from that of either p- or x-receptors. In general, d-receptors appear at later stages of neuronal development, in an anatomical distribution which is similar to that of the adult. In most brain areas in which binding appears, there is a marked increase in &receptor density during the second and third postnatal weeks. These findings are consistent with those of our membrane binding studies, in which the ratio of d:p-receptors was found to increase significantly during this time period. The ontogeny of d-receptor binding is not completely uniform in all brain areas, however. Binding in the neostriatum, olfactory tubercle and nucleus accumbens is apparent prior to binding in several other brain regions. Also, binding in the deep layers of the neocortex appears prior to binding in the superficial layers. It has been suggested that p- and d-receptors are interconverting forms of the same receptor s, and that d-receptors result from the coupling of p-receptors

38 with adenylate cyclase during ontogeny36. In several brain regions, such as the EPL of the olfactory bulb and the 'matrix' area of the caudate-putamen and olfactory tubercle, it does appear that dense b-receptor binding appears in areas of p-receptor loss. Upon closer examination, however, the present findings do not support the hypothesis of interconversion. The quantitative data reveal that the major loss of p-receptor binding in the EPL, and 'non-patch' neostriatum and olfactory tubercle, occurs well before the increase in b-receptor binding in these areas. There are several possible alternative explanations for the transient developmental appearance of p-receptors in these and other regions of the brain. In some areas, the changing pattern of receptor distribution may reflect the presence of binding sites on migrating cells, as postulated by Kent et al. 36. It may also reflect the movement of receptors from cell bodies to newly established terminal fields6s. Alternatively, the marked loss of binding in some regions may result from the degeneration of opioid receptorcontaining structural elements, or may indicate a transient expression of receptors on developing cells. Finally, in areas such as the globus pallidus and entopeduncular nucleus which contain high levels of opioid peptides in developing and adult rat brain 17'38'45"67 the decline in receptor binding may result from receptor down-regulation following the onset of synaptic activity. This latter possibility is questionable, however, in light of recent evidence that ablation of opioid peptide-containing striatal inputs does not induce opioid receptor up-regulation within these regions t . One must be cautious in interpreting developmental decrements in binding as actual losses in receptors. The apparent loss of receptor binding in some areas may be due to the 'dilution' of receptor-containing cells by the positioning of neuronal processes and glia between them. In rat brain, a large amount of glial proliferation and dendritic arborization occurs during postnatal periods 35. It has also previously been shown that myelin can cause quenching of the low activity emission generated by tritium3°. Thus, areas with a high degree of myelination may appear to 'lose' receptors as the process of myelination occurs. This may be the case in the globus pallidus and midbrain tegmentum, which possess high 'quenching coefficients' in adult brain 18. and which also show

losses of p- and r-receptor binding during development. Although such methodological problems may be eliminated by defatting tissue prior to processing, it is necessary to first cross-link the ligand to the receptor, usually by paraformaldehyde vapor exposure 29. This fixation protocol is only effective for certain ligands, such as [3H]naloxone4°. Since the specific binding of the radioligands which were employed in this study was dramatically decreased following vapor fixation and defatting (unpublished observations), this procedure was not used. It should be noted, however, that even following fixation and defatting protocols, there is relatively little [3H]naloxone binding in the globus pallidus of adult rats 29. Based on the results of pharmacological studies, it has been postulated that [Met]- and [Leu]-enkephalin are the endogenous ligands for the b-receptor 44 and dynorphin A for the r-receptor 12, while the endogenous ligand for the p-receptor is unknown. However, our present autoradiographic data do not provide evidence for a simple anatomical correspondence between any single opioid receptor type and opioid peptide system in developing or adult rat brain 17,38,45'67. When all 3 opioid peptide systems and opioid receptor types are considered together, there is a moderately high degree of overlap between peptide and receptor. Furthermore, in certain regions, such as the globus pallidus and entopeduncular nucleus which have been considered to represent areas of peptide-receptor mismatch in adult rat brain 27, we have observed a transient developmental appearance of opioid receptors. There are several possible explanations for the lack of simple correspondence between the regional distributions of each opioid receptor type and any single opioid peptide system. Endogenous opioid peptides do not exhibit absolute specificity for a given opioid receptor 12'13'15'33'44. Thus, in the absence of available 7c- or b-receptors, dynorphin A and [Leu]-enkephalin may interact with the p-receptor type 13'44. Furthermore, several recent studies have indicated that different opioid peptide products of the same propeptide precursor may have differing pharmacological selectivity profiles T M 33,68. It is therefore possible that each opioid receptor type is functionally related to different opioid peptides in different regions of the brain. The function of opiate receptors during ontogeny is still relatively unknown. Data from physiological

39 studies have provided evidence that opioid receptors are functional during the perinatal period and have the capacity to modulate developmental processes such as cellular proliferation and neuronal

velopmental role for these opioid receptor types.

death 37,5°,71. These receptors may also control certain physiological processes which are vital to the neonate, such as suckling 34 and infant-maternal bonding 31'55. Early activation of opioid receptors by exogenously administered agonists may induce longterm changes in receptor number and distribution 63, with concomitant behavioral changes TM. While ~- and x-receptors appear in certain brain regions at an early stage of development, 6-receptors appear later and in their adult distribution. Thus, the early and sometimes transient appearance of ~- and r-receptors in some brain regions may reflect a specific de-

This work was supported by National Institute of Health Grants NS 18843, NS 19319 and N I H 5 P41 RR0 1192. The authors are indebted to Drs. Sandra Loughlin, Christine Gall and James Fallon for expert anatomical advice. We also wish to thank Rick Burgoon, Glenn Chavez, Nita Patel and Sharleen Tso for excellent technical assistance, and Kathy Huettl and Cherie Jameison for manuscript preparation. Quantitative analyses were performed at the Laser Microbeam Program ( L A M P ) at the University of California, Irvine, which is a National Institute of Health Biotechnology Resource Program.

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