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Ontogeny of PCP and Sigma receptors in rat brain
Developmental Brain Research, 51 (1990) 147-152 Elsevier BRESD 51001
147
Research Reports
Ontogeny of PCP and Sigma receptors in rat brain George A. Paleos, Zan Wei Yang and James C. Byrd Developmental Neurobiology Program, Centerfor Neuroscience, Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15213 (U.S.A.) (Accepted 18 July 1989)
Key words: Phencyclidine; Brain; Rat; Phencyclidine receptor; Sigma receptor; Growth
Phencyclidine (PCP) binds with high affinity to two receptors in rat brain - - the PCP receptor and the Sigma receptor. Although both receptors are present prenatally, and their number increases postnatally, their rate of increase, compared to the increase in brain protein, is quite different, yielding distinct ontogenic profiles. Thus, PCP receptors are present on prenatal day 2 and show a further 15-fold increase by postnatal day 28. In contrast, Sigma receptors are present at their highest density during the perinatal period, and decline thereafter. The Ka of the PCP receptor for TCP remains constant throughout development, whereas the Kd of the Sigma receptor for (+)-3-(3hydroxyphenyl)-N-(l-propyi)piperidine decreases 40% postnatally. On postnatal day 6, both PCP and Sigma receptors display a pharmacological profile similar to that observed in adult animals. INTRODUCTION Phencyclidine (PCP) is a widely abused psychotomimetic c o m p o u n d 1'8'11 which has been shown to bind to specific CNS receptors 49'53. Recent studies have revealed that PCP actually binds with high affinity to two distinct receptors, the PCP receptor and the Sigma receptor ~7'18. The PCP binding site has been shown to be associated with the ion channel linked to the N M D A receptor 3' 13,14,3o,31,36,37, where PCP acts as a negative modulator of N M D A action by inhibiting N M D A - m e d i a t e d calcium influx 2'12'20'22'35"42"52. Furthermore, autoradiographic studies demonstrate a marked co-localization of N M D A receptors and [3H]TCP binding sites in rat brain 33,34, In contrast, the Sigma receptor has a higher affinity for opiates of the benzomorphan class than does the PCP receptor, to which these ligands also bind 27'54. Sigma receptors are not coupled to any other known receptor or ion channel, and their function is currently unclear 45. However, it has been recently shown that (+)-3-(3hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) is able to enhance electrically stimulated norepinephrine release in the mouse and guinea pig vas deferentia 6'47. The observation that Sigma receptor agonists are capable of blocking a non-inactivating, voltage-sensitive potassium channel in synaptosomes may provide a basis for generalizing this observation to the central nervous system 4,45. Little is known about the developmental profile of
these two PCP binding sites. A n initial study utilizing the radioligand [3H]PCP indicated that PCP binding in rat brain at the time of birth approximately equaled that of adult brain 43. However, at the time this study was done, the fact that PCP binds with high affinity to two distinct sites was unknown 27. Therefore it seemed possible that differences between the developmental profile of PCP and Sigma receptors could exist, but had previously gone undetected because of the non-selectivity of [3H]PCP. Indeed, similar problems with ligand non-specificity initially confounded the interpretation of many of the ontogenic studies of the various opiate receptors; thus, the differential rates of development of mu, delta, and kappa receptors were accurately described only after the development of ligands specific for each receptor subtype 4°'46. Accordingly, we have studied the ontogenic development of the PCP and the Sigma receptor using radioligands specific for each receptor. A preliminary report of these findings has recently been presented 39. MATERIALS AND METHODS
Membrane preparation Timed pregnant Sprague-Dawley rats (250 g) were housed in animal quarters where they were maintained on a 12-h light-dark cycle and received food and water ad libitum. On the indicated developmental days, rat pups were sacrificed by decapitation, their brains removed, weighed, and placed on ice. Brains of individual animals were pooled into 4 groups containing sufficient tissue for radioreceptor assays (see Table I). Adult samples were obtained from male Sprague-Dawley rats weighing approximately 225 g
Correspondence: J.C. Byrd, Developmental Neurobiology Program, Room E-1226, WPIC, 3811 O'Hara Street, Pittsburgh, PA 15213, U.S.A. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
148 (PoN 50). Brains were homogenized in 10 vols. of Tris-HCl (5 mM: pH 8.0; 4 °C) containing EDTA (0.1 raM) using a Brinkmann Polytron, and membranes prepared by centrifuging the crude homogenate at 18,000 rpm for 15 min. The pellet was resuspended in homogenization buffer and re-centrifuged. The resulting crude membrane fraction was resuspended in sufficient buffer to yield a final protein concentration of 5 mg/ml. Membranes were stored at -135 °C until assayed.
Binding protocol Binding studies were performed according to published protocols 5'18. In a final volume of 0.5 ml, either 0.3 mg (PrN 2 and PoN 1) or 0.4 mg (PoN 6 to adult) of membrane protein was suspended in Tris-EDTA buffer (pH 8.0), and incubated with either 1 nM [3H]TCP, which shows selective binding to the PCP receptor 48, or 1 nM (+)-[3H]3-PPP, which binds selectively to Sigma receptors 26'27. Non-specific binding was determined using 10/~M PCP for [3H]TCP binding and 1/~M haloperidol for (+)-[3H]3-PPP binding. Glutamate (100 ~uM) was added to all [3H]TCP binding assays to eliminate effects attributable to any potential developmental variations in the concentration of this excitatory amino acid, which, in conjunction with glycine, is known to affect the accessibility of NMDA-associated channel blockers (e.g. TCP; MK-801) to their binding site 24"29'41'42'44'51. However, this precaution was probably unnecessary, since the crude membrane preparations employed in these studies contained relatively high concentrations (approximately 5/~m) of both glutamate and glycine (measured using the Waters 'Pico-tag' system; data not shown); such levels of these amino acids are well above the concentrations reported to be necessary for maximal enhancement of [3H]TCP binding. Membranes were incubated for 120 min at 21 °C, and collected by vacuum filtration (Brandel filtration manifold) through glass fiber filters (Schleicher & Schuell; no. 31) pre-soaked in 0.5% polyethylenimine to reduce background binding. The filters were washed twice with ice-cold binding buffer (6-ml per wash). Total filtration/ wash time was less than I0 s. Radioactivity bound to the filters was determined by liquid scintillation spectrometry. The K d and Bronxof each of these ligands for their respective receptors were determined using both graphical Scatchard analysis as well as computer analysis by a least squares, iterative, curve-fitting program (LIGAND), with both types of analysis yielding essentially equivalent results. Previous studies in our laboratory have verified that binding under these conditions is specific (>95%), linear with respect to protein concentration, and stable within the time periods specified5.
Materials [3H]TCP (55.0 Ci/mMol) and (+)-[3H]3-PPP (99.0 Ci/mMol) were purchased from New England Nuclear (Boston, MA). TCP and PCP, as well as the stereoisomers of SKF 10,047, cyclazocine and pentazocine, were obtained from the National Institute on Drug Abuse. The stereoisomers of 3-PPP were purchased from Research Biochemicals lnc. (Natick, MA). All other compounds were of reagent grade and were obtained from Sigma Chemical Company (St. Louis, MO).
RESULTS
In Table I, the average brain weight of SpragueDawley rat pups at different gestational ages is shown. This is compared to the total protein concentration per brain. These data reveal the marked differences in the rate of increase of total brain mass as compared to total protein in the developing rat brains used in this study, and subsequently served as an important index with which to contrast the rates of PCP and Sigma receptor development.
~IABLE l
Rat brain weight and protein concentration during development Rat brain weights and protein concentrations during development. Values represent the means _+ S.E.M. The number of brains per group is indicated in parentheses.
Developmental age (days)
Brain weight (g, wet weight)
Protein concentration (mg/g brain)
PrN2 PoN 1 PoN6 PoN 9 PoN 14 PoN 21 PoN 28 Adult
0.16 _+0.01 (55) 0.22 _+ 0.02 (27) 0.56_+0.01 (80) 0.80 + (I.03 (9) 1.25 _+.+_(/.03 (5) 1.50 + 0.02 (4) 1.58 +--0.01 (4) 1.93 _+0.01 (10)
19.6+2.0(4) 26.6 + 1.6 (4) 21.1 + 1.0(4) 25.3 + 2.8 (4) 37.4 + 3.0 (4) 53.5 _+3.7 (4) 58.9 + 3.5 (4) 49.9 _+ 1.5 (6)
We then examined the pattern of developmental changes in [3H]TCP and (+)-[3H]3-PPP binding to PCP and Sigma receptors, respectively. First, a broad developmental binding profile covering a wide age range was obtained by determining the amount of each radioligand (1 nM) which was specifically bound. As may be seen in Fig. 1, the two receptors showed a quite divergent pattern of change with development. TCP binding was present on prenatal day 2 and showed a sustained increase throughout postnatal development, reaching adult levels by postnatal day (PoN) 21. In contrast, (+)-[3H]3-PPP was present at slightly higher levels in the immature brain. (+)-3-PPP binding remained relatively constant from prenatal day (PrN) 2 until PoN 14, but showed a persistent slight decline thereafter. These results suggested a marked difference in the ontogenic development of PCP and Sigma receptors in rat brain. To confirm this difference, we conducted Scatchard analyses of [3H]TCP and (+)-[3H]3-PPP binding on selected pre- and postnatal days in order to
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Fig. 1. Developmental profile of [3H]TCP (0) and (+)-[3H]3-PPP (O) binding in rat brain to PCP and Sigma receptors, respectively. Specific binding was determined as described in Materials and Methods. The S.E.M. of each point was always less than 5% of the indicated value.
149 TABLE II Binding characteristics of PCP and Sigma receptors during rat brain development
Experimental values were determined in whole brain homogenates utilizing [3H]TCP and (+)-[3H]3-PPP to label PCP and Sigma receptors, respectively. Bmax and Kd values were determined as described in Materials and Methods using at least 4 independent samples per group. The S.E.M. is given in parentheses. * Indicates a significant difference (P < 0.05) as compared to adult values using Student's t-test, with P-values adjusted for multiple comparisons using the Bonferroni correction. Developmental age
PCP receptor
Sigma receptor
B,,,,~
PrN2 PoN 1 PoN6 PoN28 Adult
Kj
Bmax
Ka
fmol/mg prot
pmol/g wet weight
nM
fmol/mg prot
pmol/g wet weight
nM
350 (+ 27) 621 (+ 23) 962(+31) 1819(_+47) 1865(+40)
6.9 (+ 0.49) 16.5 (+ 0.59) 20.3(+0.66) 107(+2.8) 83.1(+_2.0)
10(+0.6) 9 (+ 0.1) 9(+0.1) 8(_+0.7) 8(_+0.2)
2016(_+ 47) 1639 (+ 69) 1368(+28) 558(_+46) 494(+_19)
39.5 (+ 0.9) 43.6 (+ 1.8) 28.9(+0.6) 32.8(+2.7) 24.7(+_1.0)
38* (± 0.6) 49* (+- 3.6) 29 (+- 1.3) 27 (+3.9) 26 (+-1.6)
d e t e r m i n e w h e t h e r these changes in r e c e p t o r binding profile were the result of an alteration in the Bmax or the K d of each receptor. Table II shows that when the Bma x of T C P and ( + ) - 3 - P P P are expressed as fmol b o u n d / m g protein, there is a 75% decrease in labeling of Sigma receptors during the interval from PrN 2 to a d u l t h o o d , whereas binding to PCP receptors steadily increased. H o w e v e r , when these binding d a t a are expressed as total r e c e p t o r levels p e r g wet weight of brain tissue, it is clear that while the PCP r e c e p t o r still shows a m a r k e d increase in binding density, there is only a m o d e s t decrease ( a p p r o x i m a t e l y 25%) in the n u m b e r of Sigma sites. Thus, the n u m b e r of T C P receptors increases at a rate faster than both brain p r o t e i n as well as gross brain mass, in contrast to the n u m b e r of Sigma receptors, which increases at a rate far below that of protein content and s o m e w h a t less than that of brain mass. A n o t h e r perspective on the d e v e l o p m e n t of these two
receptors is o b t a i n e d by considering the absolute n u m b e r of receptors present at varying d e v e l o p m e n t a l ages. A s seen in Fig. 2, it is clear that the total n u m b e r of Sigma receptors continues to increase from PrN 2 to PoN 28, rather than decrease, as might be erroneously inferred from the data in Table II. This increase in r e c e p t o r n u m b e r is m a s k e d when these d a t a are expressed as a ratio of r e c e p t o r n u m b e r to either protein concentration or brain weight, since these latter two variables increase m o r e rapidly than does the absolute n u m b e r of Sigma receptors. The m a r k e d increase in the n u m b e r of total PCP receptors is also a p p a r e n t in this figure. Since PCP binds with high affinity to both PCP and Sigma receptors, it is of interest to consider the partitioning of total potential PCP binding b e t w e e n PCP and Sigma receptors during d e v e l o p m e n t . A c c o r d i n g l y , the ratio of the Bmax of these two sites is also shown in Fig. 2.
TABLE III
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~ 0 P o N 1 PoN6 PoN28 Adult Developmental Age (days)
Fig. 2. Total number of PCP (11) and Sigma (D) receptors in rat brain at various developmental ages. Absolute receptor levels were calculated from data in Tables I and II. Note that the scale of measurement for values on PoN 28 and adult brains differs from that used for the earlier development time points. Numbers above each bar represent the ratio of total PCP/Sigma binding sites, as determined from the Bma x values on each indicated day.
Pharmacological characteristics of PCP and Sigma receptors in rat pups on postnatal day 6
Values represent the Ki (nM) of the indicated ligand for the displacement of 1 nM [3H]TCP or 1 nM (+)-[3H]3-PPP from PCP and Sigma receptors, respectively, using binding assays as described in Materials and Methods. Each ligand was tested on 4 independent samples. The S.E.M. is indicated in parentheses, n.d., not determined. Ligand
PCP receptor
Sigma receptor
TCP PCP (+)-3-PPP (-)PPP (+)SKF 10,047 (-)SKF 10,047 (+)Cyclazocine (-)Cyclazocine (+)Pentazocine Haloperidol
12.0 (+ 0.4) 33. l (+_ 1.3) >25,000 n.d. 157 (_+ 9) 294 (_+ 11) n.d. n.d. 2354 (+ 247) > 10,000
n.d. n.d. 30.1 (+ 1.2) 123 (+ 9.4) 187 (_+ 36) 986 (+ 85) 249 (_+ 13) 513 (+ 19) n.d. 10.2 (+ 0.2)
150 Studies were also performed to determine if the affinities of the PCP and Sigma receptors for the site-specific radioligands used in these studies changed with development. These results are shown in Table II. The Ka of the PCP receptor for TCP clearly remained unchanged throughout development, while the affinity of the Sigma receptor for (+)-3-PPP increased modestly. Since there is a considerable degree of overlap in ligands active at both TCP and Sigma receptors in adult animals, we investigated the pharmacological profile of PCP and Sigma receptors on PoN 6, to determine whether such similarities in ligand affinity were also present in the early postnatal period. In Table II1, it may be seen that by PoN 6, the PCP receptor displayed essentially equivalent affinities for a variety of ligands compared to the adult 27. Importantly, this included the ability of the PCP receptor to discriminate the stereoisomers of SKF 10,047. Likewise, the Sigma receptor displayed a pharmacological profile quite similar to that seen in adult animals, including the ability to distinguish stereoisomers of 3-PPP, SKF 10,047, and cyclazocine, as well as demonstrating a high affinity for haloperidol. Thus, by PoN 6, both the PCP and the Sigma receptor have developed a pharmacological profile essentially equivalent to that seen in adult animals. DISCUSSION The results of this study demonstrate that the two major high affinity binding sites for PCP - - the PCP receptor and the Sigma receptor - - display quite distinct developmental profiles. PCP receptors show a progressive increase in their Bmax during postnatal development, with the greatest rate of increase occurring after PoN 14. These findings are consistent with the results of autoradiographic studies of the ontogeny of NMDA receptors 23, as well as with other studies which reveal a marked increase in the rate of development of glutamatergic synapses between day 12 and 21 in the rat hippocampus 5°. During this same time period, it has also been shown that pyramidal neurons in the CA1 area of the hippocampus become increasingly sensitive to the excitatory amino acid NMDA 19, which provides physiological evidence for the maturation of NMDA receptor systems, and consequently, of the NMDA-receptor-linked ion channels containing the PCP binding site. These findings also correspond with the time course of the development of rat brain glutamatergic systems as determined by binding studies measuring total [3H]glutamate to rat brain membranes ~5. These differences in developmental profile are also consistent with the anatomical distribution of these two receptors. PCP receptors, which have their greatest
density in the hippocampus and cerebral cortex a°'2;, increase later in postnatal development, during periods of synaptogenesis in these higher brain regions. In contrast, Sigma receptors, which are more highly concentrated in the brain stem and cerebellum, are present at their greatest density at birth, and decrease slowly thereafter, during the postnatal period of neocortical maturation. However, although their density decreases, the postnatal increase in the absolute number of Sigma receptors may reflect the appearance of hippocampal and cortical Sigma receptors, which are present at more moderate levels in these brain regions 26'27. In addition to improving our understanding of the patterns of development of the neurotransmitter systems utilizing PCP and Sigma receptors, the results of these studies may have practical implications as well. PCP is a well-known drug of abuse, and infants born to PCPabusing mothers are known to display a constellation of behavioral alterations of which autonomic hyperactivity is a prominent feature 7'16'2~. In view of the preponderance of Sigma receptors relative to PCP receptors at birth, these pharmacological effects of PCP on the newborn may be mediated through Sigma receptors rather than PCP receptors, an hypothesis consistent with the high density of these receptors in various brainstem regions controlling autonomic functions. Conversely, since NMDA receptors appear to play a crucial role in activity-dependent synaptic modifications such as long-term potentiation (LTP) 9, and early blockade of NMDA receptors prevents normal neural development and learning 1°'25'28, it is possible that postnatal exposure to PCP at later time periods may seriously disrupt normal neuronal development. The possibility of such exposure in humans is quite plausible since PCP is known to be concentrated and excreted in breast milk 38. A major unanswered question regarding the ontogeny of PCP and Sigma receptors concerns the rate of development of these two receptors in various brain regions. Autoradiographic studies should yield valuable information regarding the precise cellular populations undergoing changes in the expression of these two receptors during the postnatal period. These data, in turn, may shed further light on the functional implications of the developmental changes of PCP and Sigma receptors on neuronal functioning. It should be noted that the results and conclusions reported above differ somewhat from those of a similar study which was published following the submission of this manuscript 32. Specifically, Majewska et al. concluded that PCP receptors reached their maximal levels by postnatal day 14, and that the density of Sigma receptors does not change during postnatal development. Several explanations are possible for these discrepancies. First,
151 the rat strains used were different. Secondly, Majewska et al. used a non-selective radioligand ([3H]haloperidol) in c o n j u n c t i o n with additional blocking compounds to label the Sigma receptor. However, despite these methodological differences, examination of their data shows that in m a n y respects, their results are similar to our own, although Majewska et al. have drawn different conclusions from their data. First, these authors claim that PCP receptors have reached adult levels by PoN 14. However, the trend in their data indicates a steady increase in PCP receptors throughout the postnatal period, as do our data. Thus, it is possible that their m e a s u r e m e n t s on PoN 14 may be aberrantly high, although this observation could represent a true difference between the two different rat strains used. Secondly, Majewska et al. conclude that
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Sigma receptor levels do not change postnatally. However, this conclusion was drawn from binding levels expressed solely as fmol/mg protein and, therefore, does not provide a complete picture of receptor development, for the reasons that we have described above. Thus, the data of Majewska et ai. do not appear to contradict our own findings, despite the methodological differences between the studies. Rather, their data confirm the results which we have reported above, and which we have interpreted and discussed within a somewhat broader context. Acknowledgements. We thank John D. Fernstrom, Ph.D., for performing the amino acid measurements in the synaptic membrane preparations, Bryan L. Roth, M.D., Ph.D., for his interesting comments on this topic, and Mrs. Patricia Brandt for preparing the manuscript.
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42 43 44 45
46 47 48
49
50
51 52
53 54
from rat brain is enhanced by glycine, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7744-7748. Reynolds, I.J. and Miller, R.J., Multiple sites for the regulation of the N-methyl-D-aspartate receptor, Mol. Pharmacol.. 33 (1988) 581-584. Sircar, R. and Zukin, S.R., Ontogeny of Sigma opiate/phencyclidine-binding sites in rat brain, Life Sci., 33, Suppl. I (1983) 255-258. Snell, L.D., Morter, R.S. and Johnson, K.M., Glycine potentiates N-methyl-D-aspartate-induced [3H]TCP binding to rat cortical membranes, Neurosci. Lett., 83 (1987) 313-317. Sonders, M,S., Keana, J.F.W. and Weber, E., Phencyclidine and psychotomimetic Sigma opiates: recent insights into their biochemical and physiological sites of action, Trends Neurosci., 11 : 1 (1988) 37-40. Spain, J.W., Roth, B.L. and Coscia, C.J., Differential ontogeny of multiple opioid receptors (mu, delta and kappa), J. Neurosci., 5:3 (1985) 584-588. Vaupel, D.B. and Su, T.P., Guinea pig vas deferans preparation may contain both Sigma receptors and phencyclidine receptors, Eur. J. Pharmacol., 139 (1987) 125-128. Vignon, J., Chicheportiche, R., Chicheportiche, M., Kamenka, J.M., Geneste, P. and Lazdunski, M., [3H]TCP: a new tool with high affinity for the PCP receptor in rat brain, Brain Res., 280 (1983) 194-197. Vincent, J.P., Vignon, J., Kartalovski, B., Geneste, P., Kamenka, J.M. and Lazdunski, M., Interaction of phencyclidine ('angel dust') with a specific receptor in rat brain membranes, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4678-4682. Wolf, G., Richter, K., Schunzel, G. and Schopp, W., Histochemically demonstrable activity of phosphate-activated glutaminase in the postnatally developing rat hippocampus, Dev. Brain Res., 41 (1988) 101-108. Wong, E.H.F., Knight, A.R. and Ransom, R., Glycine modulates [3H]MK-801 binding to the NMDA receptor in rat brain, Eur. J. Pharmacol., 142 (1987) 487-488. Wroblewski, J.T., Nicoletti, F., Fadda, E. and Costa, E., Phencyclidine is a negative allosteric modulator of signal transduction at two subclasses of excitatory amino acid receptors, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 5068-5072. Zukin, S.R. and Zukin, R.S., Specific [3H]phencyclidinebinding in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5372-5376. Zukin, R.S. and Zukin, S.R., Demonstration of [3H]cyclazocine binding to multiple opiate receptor sites, Mol. Pharmacol., 20 (1981) 246-254.
147
Research Reports
Ontogeny of PCP and Sigma receptors in rat brain George A. Paleos, Zan Wei Yang and James C. Byrd Developmental Neurobiology Program, Centerfor Neuroscience, Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15213 (U.S.A.) (Accepted 18 July 1989)
Key words: Phencyclidine; Brain; Rat; Phencyclidine receptor; Sigma receptor; Growth
Phencyclidine (PCP) binds with high affinity to two receptors in rat brain - - the PCP receptor and the Sigma receptor. Although both receptors are present prenatally, and their number increases postnatally, their rate of increase, compared to the increase in brain protein, is quite different, yielding distinct ontogenic profiles. Thus, PCP receptors are present on prenatal day 2 and show a further 15-fold increase by postnatal day 28. In contrast, Sigma receptors are present at their highest density during the perinatal period, and decline thereafter. The Ka of the PCP receptor for TCP remains constant throughout development, whereas the Kd of the Sigma receptor for (+)-3-(3hydroxyphenyl)-N-(l-propyi)piperidine decreases 40% postnatally. On postnatal day 6, both PCP and Sigma receptors display a pharmacological profile similar to that observed in adult animals. INTRODUCTION Phencyclidine (PCP) is a widely abused psychotomimetic c o m p o u n d 1'8'11 which has been shown to bind to specific CNS receptors 49'53. Recent studies have revealed that PCP actually binds with high affinity to two distinct receptors, the PCP receptor and the Sigma receptor ~7'18. The PCP binding site has been shown to be associated with the ion channel linked to the N M D A receptor 3' 13,14,3o,31,36,37, where PCP acts as a negative modulator of N M D A action by inhibiting N M D A - m e d i a t e d calcium influx 2'12'20'22'35"42"52. Furthermore, autoradiographic studies demonstrate a marked co-localization of N M D A receptors and [3H]TCP binding sites in rat brain 33,34, In contrast, the Sigma receptor has a higher affinity for opiates of the benzomorphan class than does the PCP receptor, to which these ligands also bind 27'54. Sigma receptors are not coupled to any other known receptor or ion channel, and their function is currently unclear 45. However, it has been recently shown that (+)-3-(3hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) is able to enhance electrically stimulated norepinephrine release in the mouse and guinea pig vas deferentia 6'47. The observation that Sigma receptor agonists are capable of blocking a non-inactivating, voltage-sensitive potassium channel in synaptosomes may provide a basis for generalizing this observation to the central nervous system 4,45. Little is known about the developmental profile of
these two PCP binding sites. A n initial study utilizing the radioligand [3H]PCP indicated that PCP binding in rat brain at the time of birth approximately equaled that of adult brain 43. However, at the time this study was done, the fact that PCP binds with high affinity to two distinct sites was unknown 27. Therefore it seemed possible that differences between the developmental profile of PCP and Sigma receptors could exist, but had previously gone undetected because of the non-selectivity of [3H]PCP. Indeed, similar problems with ligand non-specificity initially confounded the interpretation of many of the ontogenic studies of the various opiate receptors; thus, the differential rates of development of mu, delta, and kappa receptors were accurately described only after the development of ligands specific for each receptor subtype 4°'46. Accordingly, we have studied the ontogenic development of the PCP and the Sigma receptor using radioligands specific for each receptor. A preliminary report of these findings has recently been presented 39. MATERIALS AND METHODS
Membrane preparation Timed pregnant Sprague-Dawley rats (250 g) were housed in animal quarters where they were maintained on a 12-h light-dark cycle and received food and water ad libitum. On the indicated developmental days, rat pups were sacrificed by decapitation, their brains removed, weighed, and placed on ice. Brains of individual animals were pooled into 4 groups containing sufficient tissue for radioreceptor assays (see Table I). Adult samples were obtained from male Sprague-Dawley rats weighing approximately 225 g
Correspondence: J.C. Byrd, Developmental Neurobiology Program, Room E-1226, WPIC, 3811 O'Hara Street, Pittsburgh, PA 15213, U.S.A. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
148 (PoN 50). Brains were homogenized in 10 vols. of Tris-HCl (5 mM: pH 8.0; 4 °C) containing EDTA (0.1 raM) using a Brinkmann Polytron, and membranes prepared by centrifuging the crude homogenate at 18,000 rpm for 15 min. The pellet was resuspended in homogenization buffer and re-centrifuged. The resulting crude membrane fraction was resuspended in sufficient buffer to yield a final protein concentration of 5 mg/ml. Membranes were stored at -135 °C until assayed.
Binding protocol Binding studies were performed according to published protocols 5'18. In a final volume of 0.5 ml, either 0.3 mg (PrN 2 and PoN 1) or 0.4 mg (PoN 6 to adult) of membrane protein was suspended in Tris-EDTA buffer (pH 8.0), and incubated with either 1 nM [3H]TCP, which shows selective binding to the PCP receptor 48, or 1 nM (+)-[3H]3-PPP, which binds selectively to Sigma receptors 26'27. Non-specific binding was determined using 10/~M PCP for [3H]TCP binding and 1/~M haloperidol for (+)-[3H]3-PPP binding. Glutamate (100 ~uM) was added to all [3H]TCP binding assays to eliminate effects attributable to any potential developmental variations in the concentration of this excitatory amino acid, which, in conjunction with glycine, is known to affect the accessibility of NMDA-associated channel blockers (e.g. TCP; MK-801) to their binding site 24"29'41'42'44'51. However, this precaution was probably unnecessary, since the crude membrane preparations employed in these studies contained relatively high concentrations (approximately 5/~m) of both glutamate and glycine (measured using the Waters 'Pico-tag' system; data not shown); such levels of these amino acids are well above the concentrations reported to be necessary for maximal enhancement of [3H]TCP binding. Membranes were incubated for 120 min at 21 °C, and collected by vacuum filtration (Brandel filtration manifold) through glass fiber filters (Schleicher & Schuell; no. 31) pre-soaked in 0.5% polyethylenimine to reduce background binding. The filters were washed twice with ice-cold binding buffer (6-ml per wash). Total filtration/ wash time was less than I0 s. Radioactivity bound to the filters was determined by liquid scintillation spectrometry. The K d and Bronxof each of these ligands for their respective receptors were determined using both graphical Scatchard analysis as well as computer analysis by a least squares, iterative, curve-fitting program (LIGAND), with both types of analysis yielding essentially equivalent results. Previous studies in our laboratory have verified that binding under these conditions is specific (>95%), linear with respect to protein concentration, and stable within the time periods specified5.
Materials [3H]TCP (55.0 Ci/mMol) and (+)-[3H]3-PPP (99.0 Ci/mMol) were purchased from New England Nuclear (Boston, MA). TCP and PCP, as well as the stereoisomers of SKF 10,047, cyclazocine and pentazocine, were obtained from the National Institute on Drug Abuse. The stereoisomers of 3-PPP were purchased from Research Biochemicals lnc. (Natick, MA). All other compounds were of reagent grade and were obtained from Sigma Chemical Company (St. Louis, MO).
RESULTS
In Table I, the average brain weight of SpragueDawley rat pups at different gestational ages is shown. This is compared to the total protein concentration per brain. These data reveal the marked differences in the rate of increase of total brain mass as compared to total protein in the developing rat brains used in this study, and subsequently served as an important index with which to contrast the rates of PCP and Sigma receptor development.
~IABLE l
Rat brain weight and protein concentration during development Rat brain weights and protein concentrations during development. Values represent the means _+ S.E.M. The number of brains per group is indicated in parentheses.
Developmental age (days)
Brain weight (g, wet weight)
Protein concentration (mg/g brain)
PrN2 PoN 1 PoN6 PoN 9 PoN 14 PoN 21 PoN 28 Adult
0.16 _+0.01 (55) 0.22 _+ 0.02 (27) 0.56_+0.01 (80) 0.80 + (I.03 (9) 1.25 _+.+_(/.03 (5) 1.50 + 0.02 (4) 1.58 +--0.01 (4) 1.93 _+0.01 (10)
19.6+2.0(4) 26.6 + 1.6 (4) 21.1 + 1.0(4) 25.3 + 2.8 (4) 37.4 + 3.0 (4) 53.5 _+3.7 (4) 58.9 + 3.5 (4) 49.9 _+ 1.5 (6)
We then examined the pattern of developmental changes in [3H]TCP and (+)-[3H]3-PPP binding to PCP and Sigma receptors, respectively. First, a broad developmental binding profile covering a wide age range was obtained by determining the amount of each radioligand (1 nM) which was specifically bound. As may be seen in Fig. 1, the two receptors showed a quite divergent pattern of change with development. TCP binding was present on prenatal day 2 and showed a sustained increase throughout postnatal development, reaching adult levels by postnatal day (PoN) 21. In contrast, (+)-[3H]3-PPP was present at slightly higher levels in the immature brain. (+)-3-PPP binding remained relatively constant from prenatal day (PrN) 2 until PoN 14, but showed a persistent slight decline thereafter. These results suggested a marked difference in the ontogenic development of PCP and Sigma receptors in rat brain. To confirm this difference, we conducted Scatchard analyses of [3H]TCP and (+)-[3H]3-PPP binding on selected pre- and postnatal days in order to
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I
0
I
7
i
14
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28
Postnatal Age, days
Fig. 1. Developmental profile of [3H]TCP (0) and (+)-[3H]3-PPP (O) binding in rat brain to PCP and Sigma receptors, respectively. Specific binding was determined as described in Materials and Methods. The S.E.M. of each point was always less than 5% of the indicated value.
149 TABLE II Binding characteristics of PCP and Sigma receptors during rat brain development
Experimental values were determined in whole brain homogenates utilizing [3H]TCP and (+)-[3H]3-PPP to label PCP and Sigma receptors, respectively. Bmax and Kd values were determined as described in Materials and Methods using at least 4 independent samples per group. The S.E.M. is given in parentheses. * Indicates a significant difference (P < 0.05) as compared to adult values using Student's t-test, with P-values adjusted for multiple comparisons using the Bonferroni correction. Developmental age
PCP receptor
Sigma receptor
B,,,,~
PrN2 PoN 1 PoN6 PoN28 Adult
Kj
Bmax
Ka
fmol/mg prot
pmol/g wet weight
nM
fmol/mg prot
pmol/g wet weight
nM
350 (+ 27) 621 (+ 23) 962(+31) 1819(_+47) 1865(+40)
6.9 (+ 0.49) 16.5 (+ 0.59) 20.3(+0.66) 107(+2.8) 83.1(+_2.0)
10(+0.6) 9 (+ 0.1) 9(+0.1) 8(_+0.7) 8(_+0.2)
2016(_+ 47) 1639 (+ 69) 1368(+28) 558(_+46) 494(+_19)
39.5 (+ 0.9) 43.6 (+ 1.8) 28.9(+0.6) 32.8(+2.7) 24.7(+_1.0)
38* (± 0.6) 49* (+- 3.6) 29 (+- 1.3) 27 (+3.9) 26 (+-1.6)
d e t e r m i n e w h e t h e r these changes in r e c e p t o r binding profile were the result of an alteration in the Bmax or the K d of each receptor. Table II shows that when the Bma x of T C P and ( + ) - 3 - P P P are expressed as fmol b o u n d / m g protein, there is a 75% decrease in labeling of Sigma receptors during the interval from PrN 2 to a d u l t h o o d , whereas binding to PCP receptors steadily increased. H o w e v e r , when these binding d a t a are expressed as total r e c e p t o r levels p e r g wet weight of brain tissue, it is clear that while the PCP r e c e p t o r still shows a m a r k e d increase in binding density, there is only a m o d e s t decrease ( a p p r o x i m a t e l y 25%) in the n u m b e r of Sigma sites. Thus, the n u m b e r of T C P receptors increases at a rate faster than both brain p r o t e i n as well as gross brain mass, in contrast to the n u m b e r of Sigma receptors, which increases at a rate far below that of protein content and s o m e w h a t less than that of brain mass. A n o t h e r perspective on the d e v e l o p m e n t of these two
receptors is o b t a i n e d by considering the absolute n u m b e r of receptors present at varying d e v e l o p m e n t a l ages. A s seen in Fig. 2, it is clear that the total n u m b e r of Sigma receptors continues to increase from PrN 2 to PoN 28, rather than decrease, as might be erroneously inferred from the data in Table II. This increase in r e c e p t o r n u m b e r is m a s k e d when these d a t a are expressed as a ratio of r e c e p t o r n u m b e r to either protein concentration or brain weight, since these latter two variables increase m o r e rapidly than does the absolute n u m b e r of Sigma receptors. The m a r k e d increase in the n u m b e r of total PCP receptors is also a p p a r e n t in this figure. Since PCP binds with high affinity to both PCP and Sigma receptors, it is of interest to consider the partitioning of total potential PCP binding b e t w e e n PCP and Sigma receptors during d e v e l o p m e n t . A c c o r d i n g l y , the ratio of the Bmax of these two sites is also shown in Fig. 2.
TABLE III
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-150 ,
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:= =~ o.
~ 0 P o N 1 PoN6 PoN28 Adult Developmental Age (days)
Fig. 2. Total number of PCP (11) and Sigma (D) receptors in rat brain at various developmental ages. Absolute receptor levels were calculated from data in Tables I and II. Note that the scale of measurement for values on PoN 28 and adult brains differs from that used for the earlier development time points. Numbers above each bar represent the ratio of total PCP/Sigma binding sites, as determined from the Bma x values on each indicated day.
Pharmacological characteristics of PCP and Sigma receptors in rat pups on postnatal day 6
Values represent the Ki (nM) of the indicated ligand for the displacement of 1 nM [3H]TCP or 1 nM (+)-[3H]3-PPP from PCP and Sigma receptors, respectively, using binding assays as described in Materials and Methods. Each ligand was tested on 4 independent samples. The S.E.M. is indicated in parentheses, n.d., not determined. Ligand
PCP receptor
Sigma receptor
TCP PCP (+)-3-PPP (-)PPP (+)SKF 10,047 (-)SKF 10,047 (+)Cyclazocine (-)Cyclazocine (+)Pentazocine Haloperidol
12.0 (+ 0.4) 33. l (+_ 1.3) >25,000 n.d. 157 (_+ 9) 294 (_+ 11) n.d. n.d. 2354 (+ 247) > 10,000
n.d. n.d. 30.1 (+ 1.2) 123 (+ 9.4) 187 (_+ 36) 986 (+ 85) 249 (_+ 13) 513 (+ 19) n.d. 10.2 (+ 0.2)
150 Studies were also performed to determine if the affinities of the PCP and Sigma receptors for the site-specific radioligands used in these studies changed with development. These results are shown in Table II. The Ka of the PCP receptor for TCP clearly remained unchanged throughout development, while the affinity of the Sigma receptor for (+)-3-PPP increased modestly. Since there is a considerable degree of overlap in ligands active at both TCP and Sigma receptors in adult animals, we investigated the pharmacological profile of PCP and Sigma receptors on PoN 6, to determine whether such similarities in ligand affinity were also present in the early postnatal period. In Table II1, it may be seen that by PoN 6, the PCP receptor displayed essentially equivalent affinities for a variety of ligands compared to the adult 27. Importantly, this included the ability of the PCP receptor to discriminate the stereoisomers of SKF 10,047. Likewise, the Sigma receptor displayed a pharmacological profile quite similar to that seen in adult animals, including the ability to distinguish stereoisomers of 3-PPP, SKF 10,047, and cyclazocine, as well as demonstrating a high affinity for haloperidol. Thus, by PoN 6, both the PCP and the Sigma receptor have developed a pharmacological profile essentially equivalent to that seen in adult animals. DISCUSSION The results of this study demonstrate that the two major high affinity binding sites for PCP - - the PCP receptor and the Sigma receptor - - display quite distinct developmental profiles. PCP receptors show a progressive increase in their Bmax during postnatal development, with the greatest rate of increase occurring after PoN 14. These findings are consistent with the results of autoradiographic studies of the ontogeny of NMDA receptors 23, as well as with other studies which reveal a marked increase in the rate of development of glutamatergic synapses between day 12 and 21 in the rat hippocampus 5°. During this same time period, it has also been shown that pyramidal neurons in the CA1 area of the hippocampus become increasingly sensitive to the excitatory amino acid NMDA 19, which provides physiological evidence for the maturation of NMDA receptor systems, and consequently, of the NMDA-receptor-linked ion channels containing the PCP binding site. These findings also correspond with the time course of the development of rat brain glutamatergic systems as determined by binding studies measuring total [3H]glutamate to rat brain membranes ~5. These differences in developmental profile are also consistent with the anatomical distribution of these two receptors. PCP receptors, which have their greatest
density in the hippocampus and cerebral cortex a°'2;, increase later in postnatal development, during periods of synaptogenesis in these higher brain regions. In contrast, Sigma receptors, which are more highly concentrated in the brain stem and cerebellum, are present at their greatest density at birth, and decrease slowly thereafter, during the postnatal period of neocortical maturation. However, although their density decreases, the postnatal increase in the absolute number of Sigma receptors may reflect the appearance of hippocampal and cortical Sigma receptors, which are present at more moderate levels in these brain regions 26'27. In addition to improving our understanding of the patterns of development of the neurotransmitter systems utilizing PCP and Sigma receptors, the results of these studies may have practical implications as well. PCP is a well-known drug of abuse, and infants born to PCPabusing mothers are known to display a constellation of behavioral alterations of which autonomic hyperactivity is a prominent feature 7'16'2~. In view of the preponderance of Sigma receptors relative to PCP receptors at birth, these pharmacological effects of PCP on the newborn may be mediated through Sigma receptors rather than PCP receptors, an hypothesis consistent with the high density of these receptors in various brainstem regions controlling autonomic functions. Conversely, since NMDA receptors appear to play a crucial role in activity-dependent synaptic modifications such as long-term potentiation (LTP) 9, and early blockade of NMDA receptors prevents normal neural development and learning 1°'25'28, it is possible that postnatal exposure to PCP at later time periods may seriously disrupt normal neuronal development. The possibility of such exposure in humans is quite plausible since PCP is known to be concentrated and excreted in breast milk 38. A major unanswered question regarding the ontogeny of PCP and Sigma receptors concerns the rate of development of these two receptors in various brain regions. Autoradiographic studies should yield valuable information regarding the precise cellular populations undergoing changes in the expression of these two receptors during the postnatal period. These data, in turn, may shed further light on the functional implications of the developmental changes of PCP and Sigma receptors on neuronal functioning. It should be noted that the results and conclusions reported above differ somewhat from those of a similar study which was published following the submission of this manuscript 32. Specifically, Majewska et al. concluded that PCP receptors reached their maximal levels by postnatal day 14, and that the density of Sigma receptors does not change during postnatal development. Several explanations are possible for these discrepancies. First,
151 the rat strains used were different. Secondly, Majewska et al. used a non-selective radioligand ([3H]haloperidol) in c o n j u n c t i o n with additional blocking compounds to label the Sigma receptor. However, despite these methodological differences, examination of their data shows that in m a n y respects, their results are similar to our own, although Majewska et al. have drawn different conclusions from their data. First, these authors claim that PCP receptors have reached adult levels by PoN 14. However, the trend in their data indicates a steady increase in PCP receptors throughout the postnatal period, as do our data. Thus, it is possible that their m e a s u r e m e n t s on PoN 14 may be aberrantly high, although this observation could represent a true difference between the two different rat strains used. Secondly, Majewska et al. conclude that
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Sigma receptor levels do not change postnatally. However, this conclusion was drawn from binding levels expressed solely as fmol/mg protein and, therefore, does not provide a complete picture of receptor development, for the reasons that we have described above. Thus, the data of Majewska et ai. do not appear to contradict our own findings, despite the methodological differences between the studies. Rather, their data confirm the results which we have reported above, and which we have interpreted and discussed within a somewhat broader context. Acknowledgements. We thank John D. Fernstrom, Ph.D., for performing the amino acid measurements in the synaptic membrane preparations, Bryan L. Roth, M.D., Ph.D., for his interesting comments on this topic, and Mrs. Patricia Brandt for preparing the manuscript.
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