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Perinatal development of the endorphin- and enkephalin- containing systems in the rat brain
Brain Research, 179 (1979) 93-101
93
© Elsevier/North-Holland BiomedicalPress
PERINATAL DEVELOPMENT OF THE ENDORPHIN- AND ENKEPHALINCONTAINING SYSTEMS IN THE RAT BRAIN*
ALEJANDRO BAYON, WILLIAMJ. SHOEMAKER,FLOYD E. BLOOM,ALICE MAUSSand ROGER GUILLEMIN ,4. II. Davis Centerfor Behavioral Neurobiology and The Laboratoriesfor Neuroendocrinology, The Salk Institute, P.O. Box 1809, San Diego, Calif. 92112 (U.S.A.)
(Accepted April 12th, 1979)
SUMMARY Radioimmunoassay and microdissection procedures were used to study the perinatal development of the endorphin- and enkephalin-containing systems in the rat brain. In contrast to values reported on adult rat, endorphin levels are much higher than enkephalin levels on embryonic day 16. The highest endorphin values are found in the diencephalon, midline telencephalon and medulla-midbrain regions. Perinatally, enkephalin content increases at a faster rate than endorphin in all brain regions, producing a marked drop of the endorphin/enkephalin ratios. Between postnatal days 6 and 25, both endorphin and enkephalin levels increase, approaching their adult distribution pattern. No correlation was found between regional distributions or rates of increase of endorphin and enkephalin in any of these developmental stages, suggesting that the two peptide systems develop independently from each other. INTRODUCTION The endogenous opioid peptides share pharmacological properties and a common N-terminal sequence of the C-terminal fragment (61-91) of fl-lipotropin (flLPH) 6,a,1°. Peptidases present in brain can cleave this fragment, fl-endorphin, into the opioids ?-endorphin (61-77, fl-LPH), a-endorphin (61-76, fl-LPH) and methionine 5enkephalin (61-65, fl-LPH)I, 16. Nevertheless, both specific radioimmunoassay and immunohistochemical studies2,8,11,17,19 indicate that the endorphin- and enkephalincontaining systems exist in separate neuroanatomical networks in the brain. This separation cannot be accounted for by a differential distribution of the pentapeptide LeuS-enkephalin a since its distribution roughly parallels Met~-enkephalin in all brain regions ~0. * A preliminary report o f s o m e o f this w o r k was presented at the 8th Annual Meetingof the Society for Neurosciences,November5-9, 1978,St. Louis, Mo., U.S.A.
94 Since study of the developmental patterns of this differential distribution could provide better insight into relations between the adult systems, we have investigated both the late prenatal and early postnatal development of the endorphin- and the enkephalin-containing systems in several regions of the rat brain. EXPERIMENTAL PROCEDURES Animals
Birth-dated Sprague-Dawley male albino rats and timed-pregnant females (timing accuracy zk 12 h) were obtained from Zivic-Miller, Pittsburgh, Pa. The age of the embryos was verified by their c r o w n - r u m p length is. The variation of this value in the same litter (8-12 animals) was less than 5 % and at least 2 litters of each age were used in this study. Results from pups of either sex were combined since no sex differences were found in the total embryonic brain endorphin or enkephalin levels. The postnatal animals were decapitated, the brain removed from the skull and freshly dissected as described below. In the prenatal experiments, the mothers were anesthetized with chloral hydrate, a laparatomy performed and the embryos individually dissected; those still in utero were kept alive, moist and warm. After decapitation, their heads were boiled for 3 min in Ringer's solution to facilitate subsequent dissection. No difference was found in total endorphin or enkephalin content between freshly dissected brains and those boiled as indicated. Dissection A basic procedure was followed to dissect under stereo microscope control the brains of both embryos and postnatal animals (Fig. 1). This procedure yielded
A
",vl\
It',W . . . .
oo
~ M ~ ~D/ ~T~ Fig. 1. Brain dissection. A: two nearly parasagittal razor blade cuts (dashed lines) are initially made from the ventral aspect of the brain, running from the tip of the olfactory tubercles through the hypothalamic commissure. From thelateral slabs thus obtained both ventral hippocampus (Hc) and striatum ~St) are blunt dissected from the cortex (CO, the amygdala remaining in this piece. B: the midline slab is processed by two frontal cuts (also shown in A; in B saggital view), one passing through the optic chiasm and the anterior commissure, and the other from the interpeduncular fossa through the caudal border of the thalamic mass. The midline cortex and dorsal hippocampus as well as the spinal cord and cerebellum were removed. The three pieces thus obtained comprise medulla-midbrain (M), diencephaIon (D), and the (preoptic) midline telencephalic structures (I"). The schematic drawings although based in the postnatal rat brain show features in common to late embryonic stages.
95 essentially comparable brain anatomical regions in animals from embryonic day 16 to postnatal day 25 and gave good reproducibility in protein content per area between animals.
Tissue extraction, protein determination, and radioimmunoassays After dissection brain tissue samples were boiled for 15 rain in 1.0 M acetic acid solution (2 ml per sample) and then homogenized in a Brinkmann polytron 16. One aliquot was taken for protein determination ~3 and another further processed by centrifugation at 1000 × g for 30 min. The supernatant was separated and lyophilized. The lyophilizate was redissolved in 1 ml phosphate buffer 0.015 M, pH 7.4, containing 0.1 ~ bovine serum albumin (Sigma, crystalline) and centrifuged again. Aliquots from the supernatant were subjected to the radioimmunoassays at two dilutions in duplicate, Brain regions from as many as 5 animals were pooled to exceed the minimum detection limits of the assays. The endorphin radioimmunoassay has been previously described 7 as reading the Leu14-His~7 segment of fl-endorphin; it is equally sensitive on a molar basis to /~-lipotropin and the 31K protein 14. The enkephalin radioimmunoassay ~7 uses an antiserum raised against LeuS-enkephalin that shows 3 cross-reactivity for MetS-enkephalin. Throughout this paper, the endorphin immunoreactivity and the enkephalin immunoreactivity found in the tissues are expressed in units, defined in each case as the ng of/~-endorphin or the ng of Leu~-enkephalin that would give an equivalent trace displacement in their radioimmunoassays. Due to the higher sensitivity of our enkephalin assay for LeuS-enkephalin, expressing the tissue immunoreactivity in terms of this pentapeptide will underestimate the contribution of MetS-enkephalin to the value observed (and vice versa). In the endorphin assay, the data obtained represents the total contribution of the different endorphin-like species; expressing results in terms of fl-endorphin will underestimate possible contributions of immunoreactive substances with higher molecular weight. Therefore, only relative amounts, ratios or rates of change during endorphin and enkephalin development have been used to draw conclusions. RESULTS
Early detectable levels of endorphin and enkephalin On embryonic day 16, brain endorphin and enkephalin values are much different han the levels in comparable region in the postnatal rat (Table I). At this developmental stage the highest endorphin levels are already found in the regions that have been shown to contain the endorphin neuronal system in the adult animal: diencephalon, preoptic midline telencephalon, and midbrain 2,17. All the regions studied showed endorphin levels that represent more than 1 ~ of the values found in the postnatal day 25 weanling rat. In contrast, enkephalin distribution at prenatal day 16 does not correlate with that found in the adult; and levels of enkephalin represent a much lower percentage of the postnatal day 25 values in all regions, except the substantial amounts present in medulla and midbrain. In additional experiments, endorphin immunoreactivity, but not enkephalin immunoreactivity, could be detected in the embryonic day 13 rat brain.
96 TABLEI Regional distribution of endorphin immunoreactivity, enkephalin immunoreactivity, and protein in the 16th embryonic day rat brain
The immunoreactivity levels are expressed in endorphin and enkephalin units as defined in Methods, and as the percentage of 25-day-old rat brain regions. Similarly, tissue protein is expressed as rag/tissue piece and as percentage of PN-25. Constituents of each brain region are given in Experimental Procedures. Brain region
Medulla and midbrain (M) Diencephalon (D) Midline telencephalon (T) Striatum (St) Ventral hippocampus (He) Cortex and amygdala (Ct)
Endorphin
Enkephalin
Protein
Units*
% of
Units
% of
mg
Tissue
PN-25 value
Tissue
PN-25 value
Tissue
% of PN-25 value
0.315 ± 0.045 0.231 ± 0.060 0.266 4- 0.052 0.086 ± 0.007 0.130 ± 0.040 0.140 4- 0.007
3.38 1.37 2.32 4.72 8.66 1.64
0.106 ± 0.012 ± 0.006 ± 0.008 ± 0.003 ± 0.014 i
1.70 5- 0.02 1.15 ± 0.04 0.33 ± 0.02 0.23 ~ 0.04 0.25 ± 0.05 1.81 ± 0.19
6.72 5.78 1.72 1.64 1.97 4.12
0.025 1.92 0.001 0.22 0.001 0.08 0.002 0.11 0.0003 0.30 0.001 0.27
* Mean of 3 independent determinations + standard error of the mean. Each determination was performed at two sample dilutions in duplicate. Each sample was obtained by pooling a brain region from 5 animals; results are expressed per brain region for one animal.
Prenatal and per±natal development
The subsequent regional development of both the endorphin and the enkephalin systems appears to proceed along different time courses (Fig. 2). The absolute amounts of both endorphin and enkephalin as well as tissue protein, increase consistently between embryonic day 16 and postnatal day 25 in all 6 regions studied (see Fig. 4). Consequently, the almost general drop in endorphin concentration observed between embryonic days 16 and 20 (in some instances extending to postnatal day 6) indicates that the endorphin fractional increase during this period is lower than that of the tissue protein. In contrast, enkephalin concentration tends to increase or remain constant during this same developmental period, showing that its fractional increase is as high or higher than that of the tissue and considerably higher than that of endorphin. Additional information is obtained comparing the average increase of endorphin or enkephalin absolute content/day in the different brain regions across defined developmental intervals (Fig. 4). The absolute rates of increase of endorphin undergo only a modest increment, if any, between the prenatal and per±natal periods. In contrast, the absolute rates of increase of enkephalin immunoreactivity, which are much lower than those of endorphin before birth, increase several-fold for the per±natal epoch (except for the medulla-midbrain region). The differences between the rates of increase of endorphin and enkephalin are reflected in a marked but gradual decrease in the endorphin/enkephalin ratios during the prenatal and (with the exception of the medulla and midbrain) per±natal periods (Fig. 3).
97 0.5
,,=,
l
,1 ,[°-- /t
Medulla + Mldbrain
Striotum
o.,t
o
,o
'0!2
!o
~
t
25
DAYS
i
J
Z
_.~t =
° ~ .~
0.5 "Cortex + Amygdolo
o-~, 26' t
~ DAYS
~']50
2~
BIRTH
BIRTH
Fig. 2. Regional patterns of the early development of the brain endorphin and cnkephalin systems expressed on a protein weight basis. The 6 panels show the data from each of the brain regions dissected as described in Methods. The developmental stages in days are indicated in the abscissa. On the ordinate, the left-hand side shows the concentrationsofendorphin ( - - © - - ) and ofenkephalin ( - - 0 - - ) expressed in units (s¢¢ experimental procedures) per mg of tissue protein; the right-hand side shows total tissue protein (--F-I--) expressed in rag. The values shown are the mean of 3-6 determinations. The bars represent the standard error of the mean (not shown when smaller than the symbol).
3"
40
"
'
i
"
t'
Io DAYS
zo
Fig. 3. Regional ¢ndorphin/enkephalin immunoreactivity ratios during early developmental stages. The abscissa show the developmental stages in days {from left to right: embryonic days 16 and 20, birth is indicated by an arrow, and postnatal days 6 and 25). The ratios ofendorphin units vs enkephalin units were calculated from individual samples. The means of 3-6 independent values =Estandard error of the mean are represented in the ordinates for the 6 brain regions defined in methods: medulla and midhrain (--©--), diencephalon (O), telencephalon (midline) ([]), striatum (1), hippocampus (ventral) {A) and cortex and amygdala ( ~ .
98 0.8
o
0.6
z z
0.4
n~ ~ 0.2
~ 0.4 I~ 0.2
,,,
o
obc
abc obc abe o bc abc I I I I ~, :I, I i I Med.+ Dienc. Telenc. Striat. Hlppoc. Cortex. Midbr. Med. + Amyg.
DEVELOPMENTAL EPOCHS:
o) ED 1 6 - ED20 b) E D 2 0 - P N 6 c) PN6 - P N 2 5
Fig. 4. Regional rates of increase of endorphin and enkephalin immunoreactivity during three early periods of development. The rates of increase (ordinates) were calculated as the total change in mean value of endorphin or enkephalin units during each developmental period, divided by the number of days in that period the abscissa indicates the 6 brain regions studied (see Experimental Procedures), the rates corresponding to each of three arbitrarily defined developmental periods: a, prenatal, from embryonic day 16 to 20; b, perinatal, from embryonic day 20 to postnatal day 6; c, postnatal, from postnatal day 6 to 25.
Postnatal development During the postnatal period from days 6 to 25, both endorphin and enkephalin concentrations increase in all brain regions except the striatum (Fig. 2). Here, there is a continuous drop in the concentration of endorphin until the low adult values are attained, indicating a low rate of increase of endorphin immunoreactivity with respect to the increase of protein only in this region. Between the perinatal and the postnatal epochs the absolute rates of increase of endorphin immunoreactivity show a several-fold increase in the diencephalon and medial telencephalon (Fig. 4). Interestingly, in the medulla-midbrain region there seems to be a steady increase in rate throughout the developmental process. The absolute rate of increase of enkephalin immunoreactivity is also higher during the period of postnatal development than during earlier periods, mainly in the medial telencephalon but also in diencephalon, striatum and the cortex-amygdala tissue. DISCUSSION Our results indicate that as early as embryonic day 16, the regional distribution of endorphin already indicates the adult pattern. Since only trace amounts of endorphin immunoreactivity can be detected in the embryonic day 13 brain, the
99 neuronal system containing endorphin presumably begins to differentiate during this period of ontogeny. This process seems to occur in parallel to that of catecholaminecontaining systems in the brain stem, detected as early as embryonic days 13 and 14, and which display at embryonic day 15 the basic adult pattern 15,1s. Interestingly, immunocytochemical studies have shown the presence of endorphin immunoreactive cells in the brain of the 1lth week human fetus 4. Also, opiate receptor binding has been found in the embryonic day 14 rat forebrain 5. In contrast, enkephalin immunoreactivity was not detectable in the embryonic day 13 rat brain and its levels are still very low 3 days later, with a regional distribution unlike that found in the adult animal. This suggests that, in general, enkephalin systems are at this time in a much less advanced stage of differentiation than the endorphin systems. This concept is in agreement with the observation that small local interneurons like the enkephalin containing cells frequently migrate and differentiate later than the larger neurons forming neural pathways over long distances, as is the case of those containing endorphin. The relatively high levels of enkephalin in the medulla-midbrain region could be an exception to this concept, and be in accordance with the idea that phylogeneticaUy older structures frequently develop earlier than the cephalic end of the neural tube 10. During the following prenatal period of development between embryonic days 16 and 20, the levels of enkephalin increase at a higher fractional rate than the endorphin content. Yet, this high fractional rate of increase is associated with only small increments in absolute amounts of enkephalin in comparison to the increases in endorphin content during this same period (Fig. 4). This fast development of the enkephalin system is analogous to that observed for endorphin between embryonic days 13 and 16, and shows a measurable delay in onset of enkephalin development with respect to that of endorphin. During the perinatal period the rates of both fractional and the absolute increase of enkephalin are in general several fold higher than those observed in the prenatal period, indicating that the initial burst of increase marking the onset of its development extends to the perinatal epoch. The extended developmental delay of the enkephalin-containing systems with respect to those containing endorphin is reflected in the gradual and continuous decrease of the endorphin/enkephalin ratios during the period between embryonic day 16 and postnatal 6. The development of both peptide systems in the medulla-midbrain region is extended and steadier, at variance with the forebrain pattern. In the period of postnatal development between days 6 to 25, both endorphin and enkephalin undergo a marked increment in their rates of absolute increase and grow at a faster fractional rate than the tissue protein - - except for the already mentioned decrease in endorphin concentration in the striatum. Remarkably, there is a rank order correlation between the value of the regional absolute rates of increase of endorphin in this period, and the levels of endorphin in the same regions in the 25 day old weanling rat (or in the adult animal). Similarly, the regions showing higher rates of increase in absolute amounts of enkephalin are the regions with the higher total enkephalin content at maturity. Also, the endorphin/enkephalin ratios show a
100 tendency to stabilize during this period. Since neuron production in the rat is largely prenatal 3, the features of these early postnatal developmental processes can be interpreted as reflecting neuronal growth, formation of new fibers and synapses, processes that take place mainly during this period 10. Studies of the ontogenetic development of opiate binding sites support this concept, showing that the rate of appearance of opiate binding sites in the rat is higher between mid-foetal stage and postnatal day 21, decreasing thereafter until adulthoodL Although not shown, we have analyzed these data for possible developmental relationships between the endorphin- and the enkephalin-containing systems. We could find no correlations between the endorphin and the enkephalin regional distribution at any of the 4 developmental time-points we studied. We also looked for dynamic relationships in the development of the two systems. No correlation was found between the rate of change of endorphin and the rate of change of enkephalin when comparing different periods of development in the same brain region. Furthermore there was no biologically meaningful correlation between these rates of increase when different brain regions were compared during the same developmental period. Altogether our data are consistent with the view that the endorphin- and the enkephalin-containing systems are independent from each other, not only in the relatively stable adult brain but also very early during the dynamic developmental process. Nevertheless, the general qualitative observation that enkephalin systems develop after those containing endorphin, with the peculiarity that endorphin regional concentrations drop as enkephalin ones tend to rise, indicates the possibility that enkephalin-producing peptidase systems could appear early during development differentiating certain endorphin-like systems into enkephalin ones. ACKNOWLEDGEMENTS We thank Ms. Viveca Andersson for excellent technical assistance. The synthetic peptides used in the radioimmunoassays were prepared by Dr. N. Ling. This work was supported by Grant DA-01785 from the National Institute on Drug Abuse. A.B. was supported by a fellowship from the Scholl Foundation. REFERENCES 1 Austen, B. M., Smyth, D. G. and Snell, C. R., 7-Endorphin, a-endorphin and MetS-enkephalin are formed extracellularly from lipotropin C fragment, Nature (Lond.), 269 (1977) 619-621. 2 Bloom, F. E., Battenberg, E., Rossier, J'., Ling, N. and Guillemin, R., Neurons containing flendorphin in rat brain exist separately from those containing enkephalin: immunocytochemical studies, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1591-1595. 3 Brasel, ~I. A., Ehrenkranz, R. A. and Winick, M., DNA polymerase activity in rat brain during ontogeny, Develop. Biol., 23 (1970)424-432. 4 Bugnon, C. et al., l~tude des Neurones lmmunoreactifs a un Immunserum Anti-[3-Endorphine chez le Foetus Humain et l'Hornrne Adult, Colloque de Neuroendocrinologie Explt., Geneve, 1978. 5 Clendennon, N. J., Petraitis, M. and Simon, E. J., Ontological development of opiate receptors in rodent brain, Brain Research, 118 (1976) 157-160. 6 Guillemin, R., Ling, N. and Burgus, R., Endorphins, peptides d'origine hypothaiamique et neurohypophysaire a activite morphino mimetique. Isolement et structure moleculaire de ra-endorphine, C.R. Acad. Sci. (Paris) Set. D., 282 (1976) 783-785.
101 7 Ouillemin, R., Ling, N. and Vargo, T. M., Radioimmunoassays for a-endorphin and fl-endorphin, Biochem. Biophys. res. Commun., 77 (1977) 361-366. 8 HSkfelt, T., Elde, R., 1ohansson, D., Terenius, L. and Stein, L., The distribution of enkephalinimmunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 9 Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. and Morris, H. R., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 258 (1975) 577-579. 10 J'acobson, M., Developmental Neurobiology, Holt, Rinehart and Winston, New York, 1970, 465 pp. 11 Lewis, R. V., Stein, S., Gerber, L. D., Rubinstein, M. and Udenfriend, S., High molecular weight opioid-containing proteins in striatum, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 4021-4023. 12 Li, C. H., Barnafi, L., Chretien, M. and Chung, D., Isolation and amino acid sequences of fl-LPH from sheep pituitary glands, Nature (Lond.), 208 (1965) 1093-1094. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 14 Mains, R., Eiper, E. and Ling, N., Common precursor to corticotropins and endorphins. Proc. nat. ,4cad. Sci. (Wash.), 74 (1977) 3014-3018. 15 Olson, L. and Seiger, A., Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Gesch., 137 (1972) 301-316. 16 Rossier, J., Bayon, A., Vargo, T. M., Ling, N., Guillemin, R. and Bloom, F. E., Radioimmunoassay of brain peptides: evaluation of a methodology for the assay of fl-endorphin and enkephalins, Life Sci., 21 (1977) 847-852. 17 Rossier, J., Vargo, T. M., Minick, S., Ling, N., Bloom, F. E. and Guillemin, R., Regional dissociation of fl-endorphin and enkephalin contents in rat brain and pituitary, Proc. nat. ,4cad. Sci. (Wash.), 74 (1977) 5162-5165. 18 Schlumpf, M., Shoemaker, W. J. and Bloom, F. E., Inncrvation of embryonic rat cerebral cortex by catecholamine-containing fibers, J. comp. Neurol., (1979) in press. 19 Watson, S..l., Akil, H., Richard, C. W. and Barchas, J. D., Evidence for two separate opiate peptide neuronal systems, Nature (Lond.), 275 (1978) 226-228. 20 Yang, H.-Y., Hong, J. S. and Costa, E., Regional distribution of Leu- and Met-enkephalin in rat brain, Neuropharmacology, 16 (1977) 303-307.
93
© Elsevier/North-Holland BiomedicalPress
PERINATAL DEVELOPMENT OF THE ENDORPHIN- AND ENKEPHALINCONTAINING SYSTEMS IN THE RAT BRAIN*
ALEJANDRO BAYON, WILLIAMJ. SHOEMAKER,FLOYD E. BLOOM,ALICE MAUSSand ROGER GUILLEMIN ,4. II. Davis Centerfor Behavioral Neurobiology and The Laboratoriesfor Neuroendocrinology, The Salk Institute, P.O. Box 1809, San Diego, Calif. 92112 (U.S.A.)
(Accepted April 12th, 1979)
SUMMARY Radioimmunoassay and microdissection procedures were used to study the perinatal development of the endorphin- and enkephalin-containing systems in the rat brain. In contrast to values reported on adult rat, endorphin levels are much higher than enkephalin levels on embryonic day 16. The highest endorphin values are found in the diencephalon, midline telencephalon and medulla-midbrain regions. Perinatally, enkephalin content increases at a faster rate than endorphin in all brain regions, producing a marked drop of the endorphin/enkephalin ratios. Between postnatal days 6 and 25, both endorphin and enkephalin levels increase, approaching their adult distribution pattern. No correlation was found between regional distributions or rates of increase of endorphin and enkephalin in any of these developmental stages, suggesting that the two peptide systems develop independently from each other. INTRODUCTION The endogenous opioid peptides share pharmacological properties and a common N-terminal sequence of the C-terminal fragment (61-91) of fl-lipotropin (flLPH) 6,a,1°. Peptidases present in brain can cleave this fragment, fl-endorphin, into the opioids ?-endorphin (61-77, fl-LPH), a-endorphin (61-76, fl-LPH) and methionine 5enkephalin (61-65, fl-LPH)I, 16. Nevertheless, both specific radioimmunoassay and immunohistochemical studies2,8,11,17,19 indicate that the endorphin- and enkephalincontaining systems exist in separate neuroanatomical networks in the brain. This separation cannot be accounted for by a differential distribution of the pentapeptide LeuS-enkephalin a since its distribution roughly parallels Met~-enkephalin in all brain regions ~0. * A preliminary report o f s o m e o f this w o r k was presented at the 8th Annual Meetingof the Society for Neurosciences,November5-9, 1978,St. Louis, Mo., U.S.A.
94 Since study of the developmental patterns of this differential distribution could provide better insight into relations between the adult systems, we have investigated both the late prenatal and early postnatal development of the endorphin- and the enkephalin-containing systems in several regions of the rat brain. EXPERIMENTAL PROCEDURES Animals
Birth-dated Sprague-Dawley male albino rats and timed-pregnant females (timing accuracy zk 12 h) were obtained from Zivic-Miller, Pittsburgh, Pa. The age of the embryos was verified by their c r o w n - r u m p length is. The variation of this value in the same litter (8-12 animals) was less than 5 % and at least 2 litters of each age were used in this study. Results from pups of either sex were combined since no sex differences were found in the total embryonic brain endorphin or enkephalin levels. The postnatal animals were decapitated, the brain removed from the skull and freshly dissected as described below. In the prenatal experiments, the mothers were anesthetized with chloral hydrate, a laparatomy performed and the embryos individually dissected; those still in utero were kept alive, moist and warm. After decapitation, their heads were boiled for 3 min in Ringer's solution to facilitate subsequent dissection. No difference was found in total endorphin or enkephalin content between freshly dissected brains and those boiled as indicated. Dissection A basic procedure was followed to dissect under stereo microscope control the brains of both embryos and postnatal animals (Fig. 1). This procedure yielded
A
",vl\
It',W . . . .
oo
~ M ~ ~D/ ~T~ Fig. 1. Brain dissection. A: two nearly parasagittal razor blade cuts (dashed lines) are initially made from the ventral aspect of the brain, running from the tip of the olfactory tubercles through the hypothalamic commissure. From thelateral slabs thus obtained both ventral hippocampus (Hc) and striatum ~St) are blunt dissected from the cortex (CO, the amygdala remaining in this piece. B: the midline slab is processed by two frontal cuts (also shown in A; in B saggital view), one passing through the optic chiasm and the anterior commissure, and the other from the interpeduncular fossa through the caudal border of the thalamic mass. The midline cortex and dorsal hippocampus as well as the spinal cord and cerebellum were removed. The three pieces thus obtained comprise medulla-midbrain (M), diencephaIon (D), and the (preoptic) midline telencephalic structures (I"). The schematic drawings although based in the postnatal rat brain show features in common to late embryonic stages.
95 essentially comparable brain anatomical regions in animals from embryonic day 16 to postnatal day 25 and gave good reproducibility in protein content per area between animals.
Tissue extraction, protein determination, and radioimmunoassays After dissection brain tissue samples were boiled for 15 rain in 1.0 M acetic acid solution (2 ml per sample) and then homogenized in a Brinkmann polytron 16. One aliquot was taken for protein determination ~3 and another further processed by centrifugation at 1000 × g for 30 min. The supernatant was separated and lyophilized. The lyophilizate was redissolved in 1 ml phosphate buffer 0.015 M, pH 7.4, containing 0.1 ~ bovine serum albumin (Sigma, crystalline) and centrifuged again. Aliquots from the supernatant were subjected to the radioimmunoassays at two dilutions in duplicate, Brain regions from as many as 5 animals were pooled to exceed the minimum detection limits of the assays. The endorphin radioimmunoassay has been previously described 7 as reading the Leu14-His~7 segment of fl-endorphin; it is equally sensitive on a molar basis to /~-lipotropin and the 31K protein 14. The enkephalin radioimmunoassay ~7 uses an antiserum raised against LeuS-enkephalin that shows 3 cross-reactivity for MetS-enkephalin. Throughout this paper, the endorphin immunoreactivity and the enkephalin immunoreactivity found in the tissues are expressed in units, defined in each case as the ng of/~-endorphin or the ng of Leu~-enkephalin that would give an equivalent trace displacement in their radioimmunoassays. Due to the higher sensitivity of our enkephalin assay for LeuS-enkephalin, expressing the tissue immunoreactivity in terms of this pentapeptide will underestimate the contribution of MetS-enkephalin to the value observed (and vice versa). In the endorphin assay, the data obtained represents the total contribution of the different endorphin-like species; expressing results in terms of fl-endorphin will underestimate possible contributions of immunoreactive substances with higher molecular weight. Therefore, only relative amounts, ratios or rates of change during endorphin and enkephalin development have been used to draw conclusions. RESULTS
Early detectable levels of endorphin and enkephalin On embryonic day 16, brain endorphin and enkephalin values are much different han the levels in comparable region in the postnatal rat (Table I). At this developmental stage the highest endorphin levels are already found in the regions that have been shown to contain the endorphin neuronal system in the adult animal: diencephalon, preoptic midline telencephalon, and midbrain 2,17. All the regions studied showed endorphin levels that represent more than 1 ~ of the values found in the postnatal day 25 weanling rat. In contrast, enkephalin distribution at prenatal day 16 does not correlate with that found in the adult; and levels of enkephalin represent a much lower percentage of the postnatal day 25 values in all regions, except the substantial amounts present in medulla and midbrain. In additional experiments, endorphin immunoreactivity, but not enkephalin immunoreactivity, could be detected in the embryonic day 13 rat brain.
96 TABLEI Regional distribution of endorphin immunoreactivity, enkephalin immunoreactivity, and protein in the 16th embryonic day rat brain
The immunoreactivity levels are expressed in endorphin and enkephalin units as defined in Methods, and as the percentage of 25-day-old rat brain regions. Similarly, tissue protein is expressed as rag/tissue piece and as percentage of PN-25. Constituents of each brain region are given in Experimental Procedures. Brain region
Medulla and midbrain (M) Diencephalon (D) Midline telencephalon (T) Striatum (St) Ventral hippocampus (He) Cortex and amygdala (Ct)
Endorphin
Enkephalin
Protein
Units*
% of
Units
% of
mg
Tissue
PN-25 value
Tissue
PN-25 value
Tissue
% of PN-25 value
0.315 ± 0.045 0.231 ± 0.060 0.266 4- 0.052 0.086 ± 0.007 0.130 ± 0.040 0.140 4- 0.007
3.38 1.37 2.32 4.72 8.66 1.64
0.106 ± 0.012 ± 0.006 ± 0.008 ± 0.003 ± 0.014 i
1.70 5- 0.02 1.15 ± 0.04 0.33 ± 0.02 0.23 ~ 0.04 0.25 ± 0.05 1.81 ± 0.19
6.72 5.78 1.72 1.64 1.97 4.12
0.025 1.92 0.001 0.22 0.001 0.08 0.002 0.11 0.0003 0.30 0.001 0.27
* Mean of 3 independent determinations + standard error of the mean. Each determination was performed at two sample dilutions in duplicate. Each sample was obtained by pooling a brain region from 5 animals; results are expressed per brain region for one animal.
Prenatal and per±natal development
The subsequent regional development of both the endorphin and the enkephalin systems appears to proceed along different time courses (Fig. 2). The absolute amounts of both endorphin and enkephalin as well as tissue protein, increase consistently between embryonic day 16 and postnatal day 25 in all 6 regions studied (see Fig. 4). Consequently, the almost general drop in endorphin concentration observed between embryonic days 16 and 20 (in some instances extending to postnatal day 6) indicates that the endorphin fractional increase during this period is lower than that of the tissue protein. In contrast, enkephalin concentration tends to increase or remain constant during this same developmental period, showing that its fractional increase is as high or higher than that of the tissue and considerably higher than that of endorphin. Additional information is obtained comparing the average increase of endorphin or enkephalin absolute content/day in the different brain regions across defined developmental intervals (Fig. 4). The absolute rates of increase of endorphin undergo only a modest increment, if any, between the prenatal and per±natal periods. In contrast, the absolute rates of increase of enkephalin immunoreactivity, which are much lower than those of endorphin before birth, increase several-fold for the per±natal epoch (except for the medulla-midbrain region). The differences between the rates of increase of endorphin and enkephalin are reflected in a marked but gradual decrease in the endorphin/enkephalin ratios during the prenatal and (with the exception of the medulla and midbrain) per±natal periods (Fig. 3).
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Fig. 2. Regional patterns of the early development of the brain endorphin and cnkephalin systems expressed on a protein weight basis. The 6 panels show the data from each of the brain regions dissected as described in Methods. The developmental stages in days are indicated in the abscissa. On the ordinate, the left-hand side shows the concentrationsofendorphin ( - - © - - ) and ofenkephalin ( - - 0 - - ) expressed in units (s¢¢ experimental procedures) per mg of tissue protein; the right-hand side shows total tissue protein (--F-I--) expressed in rag. The values shown are the mean of 3-6 determinations. The bars represent the standard error of the mean (not shown when smaller than the symbol).
3"
40
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'
i
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Fig. 3. Regional ¢ndorphin/enkephalin immunoreactivity ratios during early developmental stages. The abscissa show the developmental stages in days {from left to right: embryonic days 16 and 20, birth is indicated by an arrow, and postnatal days 6 and 25). The ratios ofendorphin units vs enkephalin units were calculated from individual samples. The means of 3-6 independent values =Estandard error of the mean are represented in the ordinates for the 6 brain regions defined in methods: medulla and midhrain (--©--), diencephalon (O), telencephalon (midline) ([]), striatum (1), hippocampus (ventral) {A) and cortex and amygdala ( ~ .
98 0.8
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DEVELOPMENTAL EPOCHS:
o) ED 1 6 - ED20 b) E D 2 0 - P N 6 c) PN6 - P N 2 5
Fig. 4. Regional rates of increase of endorphin and enkephalin immunoreactivity during three early periods of development. The rates of increase (ordinates) were calculated as the total change in mean value of endorphin or enkephalin units during each developmental period, divided by the number of days in that period the abscissa indicates the 6 brain regions studied (see Experimental Procedures), the rates corresponding to each of three arbitrarily defined developmental periods: a, prenatal, from embryonic day 16 to 20; b, perinatal, from embryonic day 20 to postnatal day 6; c, postnatal, from postnatal day 6 to 25.
Postnatal development During the postnatal period from days 6 to 25, both endorphin and enkephalin concentrations increase in all brain regions except the striatum (Fig. 2). Here, there is a continuous drop in the concentration of endorphin until the low adult values are attained, indicating a low rate of increase of endorphin immunoreactivity with respect to the increase of protein only in this region. Between the perinatal and the postnatal epochs the absolute rates of increase of endorphin immunoreactivity show a several-fold increase in the diencephalon and medial telencephalon (Fig. 4). Interestingly, in the medulla-midbrain region there seems to be a steady increase in rate throughout the developmental process. The absolute rate of increase of enkephalin immunoreactivity is also higher during the period of postnatal development than during earlier periods, mainly in the medial telencephalon but also in diencephalon, striatum and the cortex-amygdala tissue. DISCUSSION Our results indicate that as early as embryonic day 16, the regional distribution of endorphin already indicates the adult pattern. Since only trace amounts of endorphin immunoreactivity can be detected in the embryonic day 13 brain, the
99 neuronal system containing endorphin presumably begins to differentiate during this period of ontogeny. This process seems to occur in parallel to that of catecholaminecontaining systems in the brain stem, detected as early as embryonic days 13 and 14, and which display at embryonic day 15 the basic adult pattern 15,1s. Interestingly, immunocytochemical studies have shown the presence of endorphin immunoreactive cells in the brain of the 1lth week human fetus 4. Also, opiate receptor binding has been found in the embryonic day 14 rat forebrain 5. In contrast, enkephalin immunoreactivity was not detectable in the embryonic day 13 rat brain and its levels are still very low 3 days later, with a regional distribution unlike that found in the adult animal. This suggests that, in general, enkephalin systems are at this time in a much less advanced stage of differentiation than the endorphin systems. This concept is in agreement with the observation that small local interneurons like the enkephalin containing cells frequently migrate and differentiate later than the larger neurons forming neural pathways over long distances, as is the case of those containing endorphin. The relatively high levels of enkephalin in the medulla-midbrain region could be an exception to this concept, and be in accordance with the idea that phylogeneticaUy older structures frequently develop earlier than the cephalic end of the neural tube 10. During the following prenatal period of development between embryonic days 16 and 20, the levels of enkephalin increase at a higher fractional rate than the endorphin content. Yet, this high fractional rate of increase is associated with only small increments in absolute amounts of enkephalin in comparison to the increases in endorphin content during this same period (Fig. 4). This fast development of the enkephalin system is analogous to that observed for endorphin between embryonic days 13 and 16, and shows a measurable delay in onset of enkephalin development with respect to that of endorphin. During the perinatal period the rates of both fractional and the absolute increase of enkephalin are in general several fold higher than those observed in the prenatal period, indicating that the initial burst of increase marking the onset of its development extends to the perinatal epoch. The extended developmental delay of the enkephalin-containing systems with respect to those containing endorphin is reflected in the gradual and continuous decrease of the endorphin/enkephalin ratios during the period between embryonic day 16 and postnatal 6. The development of both peptide systems in the medulla-midbrain region is extended and steadier, at variance with the forebrain pattern. In the period of postnatal development between days 6 to 25, both endorphin and enkephalin undergo a marked increment in their rates of absolute increase and grow at a faster fractional rate than the tissue protein - - except for the already mentioned decrease in endorphin concentration in the striatum. Remarkably, there is a rank order correlation between the value of the regional absolute rates of increase of endorphin in this period, and the levels of endorphin in the same regions in the 25 day old weanling rat (or in the adult animal). Similarly, the regions showing higher rates of increase in absolute amounts of enkephalin are the regions with the higher total enkephalin content at maturity. Also, the endorphin/enkephalin ratios show a
100 tendency to stabilize during this period. Since neuron production in the rat is largely prenatal 3, the features of these early postnatal developmental processes can be interpreted as reflecting neuronal growth, formation of new fibers and synapses, processes that take place mainly during this period 10. Studies of the ontogenetic development of opiate binding sites support this concept, showing that the rate of appearance of opiate binding sites in the rat is higher between mid-foetal stage and postnatal day 21, decreasing thereafter until adulthoodL Although not shown, we have analyzed these data for possible developmental relationships between the endorphin- and the enkephalin-containing systems. We could find no correlations between the endorphin and the enkephalin regional distribution at any of the 4 developmental time-points we studied. We also looked for dynamic relationships in the development of the two systems. No correlation was found between the rate of change of endorphin and the rate of change of enkephalin when comparing different periods of development in the same brain region. Furthermore there was no biologically meaningful correlation between these rates of increase when different brain regions were compared during the same developmental period. Altogether our data are consistent with the view that the endorphin- and the enkephalin-containing systems are independent from each other, not only in the relatively stable adult brain but also very early during the dynamic developmental process. Nevertheless, the general qualitative observation that enkephalin systems develop after those containing endorphin, with the peculiarity that endorphin regional concentrations drop as enkephalin ones tend to rise, indicates the possibility that enkephalin-producing peptidase systems could appear early during development differentiating certain endorphin-like systems into enkephalin ones. ACKNOWLEDGEMENTS We thank Ms. Viveca Andersson for excellent technical assistance. The synthetic peptides used in the radioimmunoassays were prepared by Dr. N. Ling. This work was supported by Grant DA-01785 from the National Institute on Drug Abuse. A.B. was supported by a fellowship from the Scholl Foundation. REFERENCES 1 Austen, B. M., Smyth, D. G. and Snell, C. R., 7-Endorphin, a-endorphin and MetS-enkephalin are formed extracellularly from lipotropin C fragment, Nature (Lond.), 269 (1977) 619-621. 2 Bloom, F. E., Battenberg, E., Rossier, J'., Ling, N. and Guillemin, R., Neurons containing flendorphin in rat brain exist separately from those containing enkephalin: immunocytochemical studies, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1591-1595. 3 Brasel, ~I. A., Ehrenkranz, R. A. and Winick, M., DNA polymerase activity in rat brain during ontogeny, Develop. Biol., 23 (1970)424-432. 4 Bugnon, C. et al., l~tude des Neurones lmmunoreactifs a un Immunserum Anti-[3-Endorphine chez le Foetus Humain et l'Hornrne Adult, Colloque de Neuroendocrinologie Explt., Geneve, 1978. 5 Clendennon, N. J., Petraitis, M. and Simon, E. J., Ontological development of opiate receptors in rodent brain, Brain Research, 118 (1976) 157-160. 6 Guillemin, R., Ling, N. and Burgus, R., Endorphins, peptides d'origine hypothaiamique et neurohypophysaire a activite morphino mimetique. Isolement et structure moleculaire de ra-endorphine, C.R. Acad. Sci. (Paris) Set. D., 282 (1976) 783-785.
101 7 Ouillemin, R., Ling, N. and Vargo, T. M., Radioimmunoassays for a-endorphin and fl-endorphin, Biochem. Biophys. res. Commun., 77 (1977) 361-366. 8 HSkfelt, T., Elde, R., 1ohansson, D., Terenius, L. and Stein, L., The distribution of enkephalinimmunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 9 Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. and Morris, H. R., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 258 (1975) 577-579. 10 J'acobson, M., Developmental Neurobiology, Holt, Rinehart and Winston, New York, 1970, 465 pp. 11 Lewis, R. V., Stein, S., Gerber, L. D., Rubinstein, M. and Udenfriend, S., High molecular weight opioid-containing proteins in striatum, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 4021-4023. 12 Li, C. H., Barnafi, L., Chretien, M. and Chung, D., Isolation and amino acid sequences of fl-LPH from sheep pituitary glands, Nature (Lond.), 208 (1965) 1093-1094. 13 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 14 Mains, R., Eiper, E. and Ling, N., Common precursor to corticotropins and endorphins. Proc. nat. ,4cad. Sci. (Wash.), 74 (1977) 3014-3018. 15 Olson, L. and Seiger, A., Early prenatal ontogeny of central monoamine neurons in the rat: fluorescence histochemical observations, Z. Anat. Entwickl.-Gesch., 137 (1972) 301-316. 16 Rossier, J., Bayon, A., Vargo, T. M., Ling, N., Guillemin, R. and Bloom, F. E., Radioimmunoassay of brain peptides: evaluation of a methodology for the assay of fl-endorphin and enkephalins, Life Sci., 21 (1977) 847-852. 17 Rossier, J., Vargo, T. M., Minick, S., Ling, N., Bloom, F. E. and Guillemin, R., Regional dissociation of fl-endorphin and enkephalin contents in rat brain and pituitary, Proc. nat. ,4cad. Sci. (Wash.), 74 (1977) 5162-5165. 18 Schlumpf, M., Shoemaker, W. J. and Bloom, F. E., Inncrvation of embryonic rat cerebral cortex by catecholamine-containing fibers, J. comp. Neurol., (1979) in press. 19 Watson, S..l., Akil, H., Richard, C. W. and Barchas, J. D., Evidence for two separate opiate peptide neuronal systems, Nature (Lond.), 275 (1978) 226-228. 20 Yang, H.-Y., Hong, J. S. and Costa, E., Regional distribution of Leu- and Met-enkephalin in rat brain, Neuropharmacology, 16 (1977) 303-307.