- Email: [email protected]
Development of cerebral methionine-enkephalinergic neurons in rats: Some difference in Wistar-Kyoto rats and spontaneously hypertensive rats
Brain Research, 271 (1983)21-31 Elsevier
21
Development of Cerebral Methionine-Enkephalinergic Neurons in Rats: Some Difference in Wistar-Kyoto Rats and Spontaneously Hypertensive Rats KEIJI NAKAMURA and TETSUO HAYASHI
Department of Pharmacology, Nippon Roche Research Center, Kamakura - 247 (Japan) (Accepted December 7th, 1982)
Key words: methionine-enkephalin- opioid neurons - cerebral nuclei - development - spontaneously hypertensive rats
Development of methionine-enkephalin(ME) and ME receptor binding in the embryonic and neonatal rat cerebral nuclei was defined quantitatively by immunocytofluorescentand microautoradiographic methods. On embryonic days 14 and 18, ME was localized in I0 of 83 nuclei particularly in the n. amygdaloideus centralis, n. periventricularis, n. supraopticus, n. interpeduncularis, n. suprachiasmaticus, n. arcuatus and n. ambiguus. ME receptor binding was distributed in the former 4 nuclei on embryonic day 14 and additional 5 nuclei on embryonic day 18. At the day of birth both levels markedly increased in these nuclei (2-3 times) and abruptly emerged in 65 out of 73 nuclei in which ME neurons were not detectable in embryonic nuclei. The abrupt appearance in most nuclei at birth probably regulates nociceptive and non-nociceptivestimuli at birth and in the neonatal period. Both levels attained their maximum at the postnatal day 7 and gradually declined thereafter. Among the perinatal period examined, ME immunoreactivity in the tractus spinalis nervi trigemini and ME receptor binding in the n. tractus spinalis nervi trigemini were markedly lower in both SHR neonates than in corresponding Wistar-Kyoto rats. Lowered levels in both areas of neonatal SHR may be involved in central hyperreactivity and preganglionic sympathetic activation of young SHR. INTRODUCTION
The enkephalin pentapeptides 22,23,46 have a widespread distribution in the brain. The binding of radiolabeled enkephalins directly to brain membranes is described 31.57. In the adult rat brain, methionine-enkephalin (ME) levels are found to be about three47 or severaP 4 times higher than leucine-enkephalin (LE) levels. In the adult rat brain, there are regional mappings for enkephalins with the antisera to both ME and LE45,48m and with the antisera to ME with some cross-reactivity to LE 19-21. There are several reports describing ontological alterations of immunoreactive enkephalinsz,39 and synthetic [3H]opiate agonist and antagonist bindings in the rat brain regions l,z,s,9.5° and the rat whole brain homogenates9,38,57. However, until recently, selective antisera to ME for quantitative analysis of cerebral nuclei and also ME-receptor binding in nuclei had not been available 32. Thus, 0006-8993/ 83 / $03.00 © 1983 Elsevier Science Publishers B.V.
developmental alteration of immunoreactive and receptor binding levels in various cerebral nuclei might be difficult to detect. Recent functional studies suggest that endogenous enkephalins are involved in cerebral cardiovascular, control, since naloxone-reversal alteration of blood pressure and heart rate is produced by the intracisternal or intracerebral administration of metabolically stable synthetic MEs 3,4,30,40,43and putative enkephalin releasers29. In some cerebral nuclei of young and adult spontaneously hypertensive rats (SHR), there are alterations of immunoreactive ME and receptor binding levels as compared to those of normotensive Wistar- Kyoto rats (WKY) 32.33. In this report we now present evidence that ME and ME receptor binding levels are distributed in the limited nuclei of prenatal rat brains and abruptly appear in most of cerebral nuclei in the day of birth. Although there is no difference in both levels between SHR and WKY dur-
ing the prenatal period, there are lowered ME levels in the tractus spinalis nervi trigemini (Vts) and ME receptor binding in the n. tractus spinalis nervi trigemini (Vnts) of SHR at the early postnatal period. MATERIAL AND METHODS
The experiments were performed on 4 paired groups of male S H R and age-matched male normotensive W K Y raised in our animal facilities. The animals used were embryonic days 14 and 18 as estimated by vaginal plug formation, the day of birth and one week after birth. In 83 different cerebral nuclei, tracts and areas, both ME contents and ME receptor binding were measured quantitatively by autoradiograms prepared by the immunohistofluorescence antibody and by [3H]ME respectively, as described previously32'33. In brief, for measuring immunoreactive ME levels of the cerebral nuclei, the animals were sacrificed by intracardiac perfusion of icecold 4% paraformaldehyde in a buffered solution. From each frozen brain, coronal sections (25 ~m) were cut serially and processed as described previously 32-3~-45. They were then mounted on glass cover-slips and brought to reaction for 30 min at 37°C with 5-fold diluted anti-ME rabbits sera. The antiserum, a gift of Drs. Takagi and Kuraishi (Kyoto), was not cross-reactive with/~-endorphin (less than 0.04%) and with LE (0.008%)27. The treated sections were subsequently incubated for 15 min at 37 °C with fluorescence-conjugated goat antibody (20-fold diluted) against rabbit IgG (Difco). For standards, we used 8% gelatine sections containing 8 different concentrations ( ~ 1280 pmol/g) of ME. The silver granule density of the fluorescence micrographs obtained by exposure to Kodak plus X was measured with a microdensitometer (Gamma Scientific) in cerebral areas of 50 ~m diameter. For measuring MERB, the animals were sacrificed by cardiac perfusion of ice-cold 0.1% formaldehyde in a buffered solution (pH 7.4). From each frozen brain, cryostat brain sections (5 ~m) were mounted on microscope slides and
incubated for 60 min at 40"C with 4 nM of pH]ME (spec. act. of 40-60 Ci/mmol, New England Nuclear) as described previouslf :, For the non-specific binding, unlabeled ME (4~M) was further added to the incubated medium on the mounted tissues. Non-specific binding which was defined as the binding in the presence of unlabeled ME amounted to about 3% of the total binding in most nuclei. For reference standards, we used 8% gelatine slices (5 /,m) containing 9 different [3H]ME concentrations (0 1280 pmol/g). After cover-slips were dipped in a solution of Kodak NTB-2 photographic emulsion (2-fold diluted), the emulsion-coated cover slips were dried and glued onto the slides. Cover-slip and slide were clamped tightly together with a clip and kept in the light-proof condition until the exposure was complete (8-10 weeks). The autoradiographic image on the cover-slip was then developed, and the silver granule density (200 >m diameter) was measured with the microdensitometer. Brain anatomy and terminology were as described by K6nig and Klippe124 and Palkovits and Jacobowitz 3v. Antinociceptive threshold was determined by a minor modification of the hot-plate test for rats as previously described ~.53. Tests were performed in a plexiglass chamber with dimensions of 25 × 25 x 25 cm. The floor of the chamber was constantly heated at 55 m 0.3 °C by circulation of warm water. The threshold was measured by the latency to lick the forepaw or j u m p from the floor. Each animal was tested 3 times at 10 min intervals, and an average response of the last two times was calculated. This procedure eliminated any novelty influence of handling or heat exposure on test results. To avoid abdominal burning, the hot-plate test for neonates was not performed. In another test pain threshold was determined by the rat tail flick method as previously described 34. Heat from an infra-red lamp was focused on the tip of the tail of animals. The latent time to remove the tail was measured 3 times at 10 min intervals and the results of the last twice averaged. Aortic blood pressure was measured in some dams without anesthesia and restraint with a pressure transducer (Nihon Koden, MPU0.5) through a ca-
23 TABLE I
Cerebral nuclei with the presence of methionine-enkephalin-like immunoreactivity in the embryo and neonate of normotensive Wistar- Kyoto rats (WKY) and spontaneously hypertensive rats (SH R) Data ( p m o l / g ) are m e a n s __. S.E.M. for 8 animals per each group.
CerebraI n uclei
R ats
Embryo nic days 14
N.ambiguus N. interpeduncularis N. reticularis lateralis N. supraopticus N. suprachiasmaticus N. periventricularis N.arcuatus N. amygdaloideus lateralis N. amygdaloideus centralis N. amygdaloideus medialis
WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR
88 89 163 151 54 55 211 209 241 255 296 266 211 225 33 36 277 276 58 65
Neon ataI days 18
+ 5.9 ___ 6.4 ± 10.7 ± 9.5 __. 3.7 __. 4.4 ± 15.2 __. 13.4 ± 16.3 __. 20.7 +-- 22.7 + 17.4 __. 16.3 ± 13.5 --. 2.2 ± 1.9 __. 18.5 ± 17.3 +-. 3.9 +_. 4.1
123 120 236 219 62 63 258 266 318 309 336 329 274 241 42 42 334 345 82 81
0 __. 8.4 __. 8.9 ± 22.4 ± 15.6 ± 4.5 __. 4.7 __. 16.4 __. 15.9 __. 22.8 +_. 21.6 ± 22.9 + 21.7 __. 21.5 ± 20.8 __. 3.4 ± 2.5 __. 22.7 ___ 26.3 __. 3.1 __. 6.2
381 368 627 620 95 81 671 690 751 781 966 918 677 709 113 109 849 852 142 152
7 __. 26.4 __. 31.5 ± 48.2 __. 40.1 __. 7.6 ± 6.3 __. 49.2 __. 58.4 ± 57.4 __. 68.3 ___ 65.9 ± 76.5 ± 49.3 ± 51.7 __. 8.7 ± 7.8 __. 67.2 ___ 63.1 ± 10.5 __. 9.7
409 387 684 680 105 97 741 732 849 819 1,015 1,059 770 785 127 135 916 935 172 161
__. 27.6 __. 33.9 __. 51.7 ± 51.9 __. 8.6 ± 6.5 ± 57.3 ± 55.1 ± 67.4 ± 75.9 __. 84.7 __. 72.6 ± 60.5 __. 58.8 ± 7.9 __. 8.5 __. 62.7 ± 71.2 ___ 10.5 __. 11.3
T A B L E II
Cerebral nuclei with the presence of methionine-enkephalin receptor binding in the embryo and neonate of normotensive WistarKyoto rats (WK Y) and spontaneously hypertensive rats (SHR) Data ( p m o l / g ) are m e a n s ± S.E.M. for 5 animals per each group.
Cerebral nuclei
N.ambiguus N. interpeduncularis N. reticularis lateralis N.supraopticus N. suprachiasmaticus N. periventricularis N. arcuatus N. amygdaloideus centralis N. amygdaloideus medialis
Group
WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR
Embryonic days
Neonatal days
14
18
~10 ~ 10 86 __. 9.3 91 ± 8.7 ~ 10 < 10 123 ± 16.4 182 ± 14.7 < 10 ~10 186 ± 20.5 167 __. 17.8 < 10 <10 210 ± 19.6 221 ___ 21.5 ~ 10 ~10
51 55 148 163 41 46 165 182 224 241 247 236 186 167 242 266 53 48
0 ± 4.8 __. 6.3 __. 16.5 ± 18.2 ± 4.3 ± 5.2 ± 19.5 ± 19.3 __. 26.3 __. 25.8 ± 25.5 ± 24.8 __. 20.4 __. 18.3 ± 26.3 ± 28.5 ± 5.6 __. 5.1
246 263 413 427 68 59 481 446 496 525 601 638 434 411 574 583 96 89
7 __. 21.3 ___ 22.8 __. 39.6 ± 41.4 ± 6.3 ± 6.7 __. 40.9 ± 38.1 ± 41.5 ± 43.9 ± 57.4 ± 59.5 ± 40.1 ___ 32.7 ± 46.3 ± 52.6 ± 8.4 ± 9.1
315 326 502 483 81 85 549 568 589 558 675 681 478 485 669 643 103 ! 12
__. 28.1 __. 25.0 __- 42.6 ± 41.8 __.8.3 __. 7.5 __. 50.7 __. 67.5 ± 50.6 __. 48.2 ___ 61.5 __. 64.4 + 42.3 ± 45.6 ± 58.4 ± 62.8 __. 9.6 __. 9.3
24 T A B L E II1
Methionine-enkephalin-like immunoreactivity in cerebral n uclei of neonates of normotensive Wistar-- Kvoto rats ( WK Y) and spontaneous!v hypertensive rats (SHR) in which M E immunoreactivity was not detected in embryonic brains Data ( p m o l / g ) are m e a n s 4- S.E for 8 animals.
Structure
Postpartum Day 0
%change
WK Y (8) Rhombencephalon and mesencephalon N. intercalatus N.commissuralis Areapostrema N. tractus solitarii (caudal) N . o r i g i n i s n. hypoglossi N . d o r s a l i s n. vagi N. olivaris inferior N. tractus spinalis n. trigemini T r a c t u s s p i n a l i s n . trigemini N. p a r a b r a c h i a l i s dorsalis N. p a r a b r a c h i a l i s v e n t r a l i s S u b s t a n t i a n i g r u s pars c o m p a c t a S u b s t a n t i a grisea centralis
107 418 179 870 311 318 266 344 1086 1044
4- 8.4 4- 28.7 4- 13.6 4- 66.3 4- 25.4 ___ 26.5 4444-
20.5 27.6 82.5 96.3
SHR (8) 111 406 185 841 301 308 249 231 1044 1014
4- 9.9 4- 34.6 4- 16.4 4- 64.7 4- 25.3 ___ 22.6 4444-
21.7 16.5" 76.1 76.6
-33
Day 7
% change
WK Y (8)
SHR (8)
125 457 221 908 340 362
128 443 219 891 351 358
293 395 1120 1106
4- 10.8 4- 35.7 4- 15.4 4- 76.5 ___ 26.5 4- 28.1
444444-
10.7 33.2 17,5 83.6 27.7 26.4
4- 22.3 286 4- 23.5 4- 27.4 263 4- 24,2* 4- 96.9 1 1 0 2 4 - 9 1 . 4 ___ 104.2 1078 4- 83.5
506 4- 42.3
491 ___ 33.6
532 4- 46.4
510 4- 38.7
Thalamus and epithalamus N. p a r a m e d i a n u s N. c e n t r o m e d i a n u s N. d o r s o m e d i a l i s N. v e n t r o m e d i a l i s N. h a b e n u l a e m e d i a l i s N. h a b e n u l a e lateralis
105 85 89 81 142 155
444444-
7.5 6.4 7.1 5.7 10.5 8.7
98 87 92 84 151 143
444444-
7.3 6.1 6.8 6.3 8.5 11.7
115 98 103 94 150 156
107 101 97 90 156 150
4- 7.3 ___ 6.3 ___ 7.6 4- 7.1 ___ 10.4 --- 11.2
F o r m a t i o reticularis N. r a p h e o b s c u r u s N. r a p h e p a l l i d u s N.raphemagnus N. t e g m e n t i d o r s a l i s N. t e g m e n t i ventralis N. l i n e a r i s m e s e n c e p h a l i N. reticularis centralis N. reticularis gigantocellularis N. reticularis paragigantocellularis N. r u b e r m e s e n c e p h a l i N. reticularis parvocellularis N. reticularis m e d i a l i s N. reticularis dorsalis S u b s t a n t i a reticularis m e s e n c e p h a l i Z o n a incerta N. intralaminalis N. reticularis t h a l a m i
237 215 284 963 563 236 57 59 53 144 120 88 51 76 74 53 -
4- 21.3 4- 15.4 4- 18.5 4- 65.4 4- 39.5 4- 17.5 4- 4.4 4- 4.7 4- 4,1 4- 10.5 4- 8.9 4- 6.2 __. 4.3 ___ 6.5 ___ 6.2 4- 4.1
226 218 276 1016 533 248 59 55 78 149 117 95 50 79 78 51 -
4444444444444444-
16.3 17.8 21.5 73.9 38.7 16.9 5.2 4.6 4.1 10.7 8.5 7.1 4,1 7.2 5.3 4. I
266 236 324 1084 634 268 64 64 60 166 133 95 59 91 86 60
21.2 16.4 21.3 72.5 41.7 15.8 4.2 5.1 5.1 11.2 8.4 7.0 3.7 7.1 6.3 5.6
243 237 318 1088 635 281 65 65 62 158 136 97 63 87 83 62
4- 18.7 4- 18.5 4- 26.4 __. 75.2 4- 44.2 ___ 17.7 4- 5.3 --- 4.1 + 4.2 ___ 11.8 4- 9.1 4- 7.5 --- 4.3 4- 7.2 --- 4.9 4- 4.2
Hypothalamus N. p o s t e r i o r h y p o laalami N. corporis m a m m i l l a r i s medialis N. corporis m a m m i l l a r i s lateralis N. v e n t r o m e d i a l i s N. d o r s o m e d i a l i s Area lateralis h y p o t h a l a m i Area anterior h y p o t h a l a m i N. paraventricularis Median eminence lnfundibulum
108 61 56 105 99 188 176 585 65 -
__. 8.4 4- 4.7 --- 3.0 4- 9.5 4- 7.8 4- 14.3 4- 14.2 4- 44.2 4- 5.1
113 58 54 102 105 181 181 565 60 -
4- 8.5 4- 4.4 __. 3.7 __. 9.1 __. 7.6 __. 14.9 4- 15.2 4- 43.6 4- 4.2
120 75 64 116 111 205 208 647 74
__. 8.6 4- 5.9 __. 3.8 __- 8.7 __. 6.5 4- 15.8 4- 16.4 ___ 42.3 __. 4.2
119 77 63 111 118 217 215 668 75
__. 8.5 __. 5.8 __. 4.3 _4- 10.5 4- 8.7 4- 13.1 ___ 18.2 4- 40.2 4- 5.3
4- 8.4 ___ 7.6 ___ 7.6 4- 6.5 ___ 11.3 4- 13.4
4444444444444444-
~--33
25 TABLE III
(Continued)
Structure
Postpartum 56change
Day 0
WKY (8) Fasciculus medialis telencephali (caudal) Fasciculus medialis telencephali (rostral) Fasciculuslongtidunalisdorsalis Forel H Telencephalon N. amygdaloideus intercalatus N. amygdaloideus basalis pars lateralis N. amygdaloideus basalis pars medialis N. araygdaloideus corticalis Area amygdaloidea anterior N. tractus olfactorius lateralis N. proprius striae terminalis N. interstitialis striae terminalis dorsalis N. interstitialis striae terminalis ventralis N. interstitialis striae terminalis medullaris N. dorsalis septi N. medialis septi N. lateralis septi N. intermedius septi N. accumbens septi N. caudatus Tuberculum olfactorium Cortex cerebri Hippocampus dorsalis Hippocampus ventralis Gyrus dentatus Subiculum Cortex entorhinalis Cortex piriformis Cortex cingutaris Cortex frontalis
111 95 201 126
± ± ± ±
SHR (8)
8.3 7.1 16.3 9.3
65-+4.3 53 ± 3.6 55 ± 3.8 57 ± 3.9 52 ± 3.6 163 ± 11.5 751 - 62.3 769 ± 54.8 711 ± 47.6 701 -+ 46.5 77 ± 5.1 59 ± 2.8 110±7.5 64 ± 4.8 869 ± 63.2 159 ± 10.4 133 ± 9.4
71 69 80 71
± ± ± ±
5.2 4.4 6.2 5.3
Day 7
%change
WKY(8)
SHR(8)
± 7.4 ±7.6 ± 14.2 ± 10.7
114 105 230 168
± ± ±
7.0 7.6 16.4 10.3
115 96 217 157
_ 8.3 ± 8.1 __. 17.4 ± 11.3
61 ± 4 . 0 54 ± 4.2 49 ± 3.7 58 ± 4.1 54 ± 4.5 169 ± 10.2 763 ± 49.5 795 ± 61 3 745 ± 36.7 722 ± 57.3 75 ± 6.8 61 _ 5.3 115±8.1 61 ± 4.9 892 ± 66.5 153 ± 11.3 142 ± 10.8
78 58 64 70 58 190 811 860 811 824 81 64 112 74 921 190 151
+ ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.1 4.0 4.9 4.7 3.3 11.4 65.1 70.5 55.3 56.7 5.7 3.3 7.4 5.5 63.9 13.4 15.2
84 55 61 76 60 182 802 837 822 815 81 64 121 71 905 203 144
_ ± ± _ ± ± ± ± ± ± ± ± _ ± ± ±
5.7 3.7 4.8 4.9 4.6 11.5 58.2 58.1 48.5 57.6 5.5 4.4 8.3 5.9 60.8 15.6 10.3
85 74 95 81
± ± ± ±
5.1 4.8 4.9 3.7
86 75 91 84
± ± ±
5.2 4.1 6.0 5.8
115 91 190 139
69 68 85 71
± ± ± ±
4.8 5.2 6.8 5.2
* P < 0.01 compared with corresponding controls - ~ 20 pmol/g.
theter implanted chronically into the aorta near the orifice of the renal artery via the femoral artery and heart rate was triggered by blood pressure pulses36. Mean aortic blood pressures of female SHR and WKY before copulation were 178 _ 5 mm Hg (means _ S.E.M., n = 5)and 112 _ 4 mm Hg, respectively. For statistical analysis, Student's t-test was employed. RESULTS
Embryonic days 14 and 18 On embryonic days, localizations of cerebral ME and receptor binding were much more limited than those in the postnatal rat as shown below. At the embryonic developmental stage im-
munoreactive ME levels were found in 10 nuclei out of 83 cerebral nuclei and areas, particulary in the nucleus (n.) amygdaloideus centralis, n. periventricularis, n. supraopticus, n. interpeduncularis and n. suprachiasmaticus (Table I). Between embryonic days 14 and 18, ME levels increased by 20-40%. On embryonic day 14, [3H]ME receptor binding levels were found in the above former 4 nuclei and additional 5 nuclei on the embryonic day 18 (Table II). During the embryonic period there was no difference in these values between corresponding nuclei of SHR and WKY embryos (Tables I and II).
26 T A B L E IV
Methionine enkephalin receptor binding in cerebral nuclei of neonates of 14/KY and SHR in which [3H]ME receptor binding was not detected in the embryonic brains D a t a ( p m o l / g ) are m e a n s +_ S.E. for 5 animals.
Structure
Postpartum day 0
%change
day 7
% change
WKY(5)
SHR(5)
WKY(5)
SHR(5)
Rhombencephalon and mesencephalon N. intercalatus N. c o m m i s s u r a l i s Areapostrema N. tractus solitarii (caudal) N.originisn.hypoglossi N.dorsalisn.vagi N. olivaris inferior N. p a r a b r a c h i a l i s dorsalis N. parabrachialis ventralis Substantia nigra Substantia grisea centralis
65 306 129 542 210 225 748 726 386
± ± ± ± ± ±
± ± _ ± ± ±
81 351 146 628 262 281
84 374 138 592 248 293
± 33.7
71 322 116 567 215 237 792 708 398
Thalamus and epithalamus N. p a r a m e d i a n u s N.centromedianus N. d o r s o m e d i a l i s N. v e n t r o m e d i a l i s N. t r a c t u s s p i n a l i s n. trigemini T r a c t u s s p i n a l i s n . trigemini N. h a b e n u l a e medialis N. h a b e n u l a e lateralis
68 71 65 61 186 221 97 103
± ± ± ± ± ± ± ±
7.1 6.5 5.7 5.1 14.7 18.5 8.4 9.6
F o r m a t i o reticularis N. r a p h e o b s c u r u s N. r a p h e p a l l i d u s N.raphemagnus N. t e g m e n t i dorsalis p a i n N. t e g m e n t i ventralis N. l i n e a r i s m e s e n c e p h a l i N. reticularis centralis N. reticularis gigantocellularis N. reticularis paragigantocellularis N. r u b e r m e s e n c e p h a l i N. reticularis parvocellularis N. reticularis medialis N. reticularis dorsalis S u b s t a n t i a reticularis m e s e n c e p h a l i Z o n a incerta N. intralaminalis N. reticularis t h a l a m i
158 146 187 718 381 145 36 39 38 97 90 61 37 51 44 -
Hypothalamus N. posterior h y p o t h a l a m i N. corporis m a m m i l l a r i s medialis N. corporis m a m m i l a r i s lateralis N. v e n t r o m e d i a l i s N. d o r s o m e d i a l i s Arealateralishypothalami A r e a anterior h y p o t h a l a m i N. paraventricularis Median eminence lnfundibulum
5.8 25.9 11.3 48.6 20.6 19.4
5.2 21.8 10.5 53.1 18.3 21.7
_ 6.7 ___ 29.6 ± 12.5 ± 51.3 ± 25.2 ± 24.6
± ± ± ± ± ±
6.2 33.8 14.9 48.6 22.7 27.3
± 71.2 ± 60.5
853 --- 62.1 862 ± 73.0
829 ± 53.6 892 ± 65.9
± 31.3
462 ± 41.5
436 ± 38.2
60 63 72 64 132 240 91 111
± ± ± ± ± ± ± ±
6.4 5.7 5.4 6.2 10.7" 21.6 9.3 8.7
85 ± 8.4 89 ± 7.6 84±7.1 74 ± 8.3 226 ± 21.5 267 ± 25.8 105 ± 8.1 126 ± 10.2
88 ± 7.3 95 _ 8.9 85±6.4 78 ± 7.4 151 ± 24.3* 277 ± 21.8 111 ± 8.5 131 ± 9.7
± 14.5 ± 12.1 ± 15.4 ± 66.2 ± 24.3 ± 11.4 ± 4.2 ± 3.5 ± 3.0 ± 5.8 ± 7.5 __. 5.8 ± 2.4 ± 4.3 ± 4.2
167 149 176 707 352 130 30 33 35 90 81 66 31 58 47 -
± ± ± ± ± _ ± ± ± ± ± ± ± ±
13.2 13.6 16.9 68.4 31.7 12.7 2.1 2.8 3.3 8.7 8.4 6.3 3.5 5.6 3.9
189 173 211 816 452 186 51 48 42 113 104 80 48 72 53
± 16.7 _ 15.1 ___ 18.4 ± 65.0 ± 38.9 __. 18.2 ± 4.8 ± 3.2 ± 3.9 ± 10.5 ± 9.6 ± 6.5 ± 4.1 ± 6.9 ± 4.3
193 170 219 791 473 172 55 53 44 118 112 87 42 64 58
± ± ± ± ± ± ± ± ± ± ± ± ± ±
18.1 15.8 22.3 73.9 45.8 16.5 3.6 3.1 3.6 9.7 10.7 7.2 3.4 6.7 3.7
68 ± 5.4 45 ± 4.2 41 _ 4.3 65 ± 7.2 68 ± 5.4 136 ± 10.5 1 3 0 ± 11.5 403 __+ 38.7 41 ± 3.6
61 49 48 60 62 139 111 385 44
± ± _ ± ± ± ± ± ±
6.7 4.8 5.6 5.8 7.4 14.2 12.7 30.3 4.5
81 56 58 78 81 162 158 462 62
_ _ _ + + ± _
87 58 64 80 75 175 142 481 63
+ ± _ _ ± _ ± _ _
7.1 4.9 5.3 7.6 7.0 15.8 13.9 44.7 4.7
_ 65.3 ± 61.8
-29
7.5 4.1 5.2 8.0 6.4 13.3 11.8 41.3 5.2
-33
27 Table IV (Continued)
Structure
Postpartum
a~yo Fasciculus medialis telencephali (caudal) Fasciculus medialis telencephali (rostral) Fasciculus longitudinalis dorsalis Forel H Telencephalon N. amygdaloideus lateralis N. amygdaloideus intercalatus N. amygdaloideus basalis pars lateralis N. amygdaloideus basalis pars medialis N. amygdaloideus corticalis N. amygdaloidea anterior N. tractusolfactoriuslateralis N. proprius striae terminalis N. interstitialis striae terminalis dorsalis N. interstitalis striae terminalis centralis N. interstitialis striae terminalis medullaris N. dorsalis septi N. medialis septi N. lateralis septi N. intermedius septi N.accumbenssepti N. caudatus Tuberculum olfactorium Cortex cerebri Hippocampus dorsalis Hippocampus ventralis Gyrus dentatus Subiculum Cortex entorhinalis Cortex piriformis Cortex cingularis Cortex frontalis
%change
a~y7
%change
WK Y (5)
SHR (5)
WK Y (5)
SHR (5)
75 61 143 85
6.2 5.1 12.0 7.6
83 65 156 81
± ± ± ±
85 ± 71 ± 160 ± 91 ±
91 77 158 94
71 ± 7.5 48 _ 3.7 41 ± 3.6 40 ± 3.5 44 ± 3.2 38 __- 3.0 111 ± 10.4 516 ± 48.5 486 ~ 41.9 389 ± 42.1 368 ± 33.5 51 ± 3.8 42 ___3.4 85 ± 7.3 42 ± 3.3 586 ± 42.7 107 ± 6.5 86 ± 7.6
64 52 44 38 42 35 118 482 463 421 362 58 44 92 45 601 113 92
± 6.9 ± 4.5 ± 4.2 _.+ 3.1 ± 3.3 ± 4.2 ___ 10.9 ± 42.7 ± 37.5 ± 40.3 ± 34.8 ± 5.3 ± 3.9 ± 8.2 ± 4.1 ± 59.6 ± 8.5 ± 6.7
-
45 51 63 55
± ± ± ±
~ 4.1 -_- 4.5 ± 4.8 ± 4.1
5.8 4.1 11.3 6.9
7.1 7.0 11.5 7.2
84 ± 8.0 62±4.1 49 ± 4.3 51 ± 4.3 48 ± 5.0 45 ± 4.2 141 ± 12.5 623 ± 51.3 548 ± 49.2 485 - 42.3 40~ ± 41.7 74 ± 6.2 52±4.1 113___11.5 52 ± 2.6 715 ± 50.2 132 ± 8.7 103 ± 8.1
-
-
___3.8 ± 4.2 ± 5.2 ± 3.7
6.4 6.2 13.2 7.5
91 ± 7.7 66±4.9 55 ± 4.7 47 ± 3.8 55 ± 5.2 47 ± 3.6 132 - 10.6 605 ± 55.7 581 ± 47.0 472 ± 40.5 422 ± 38.8 70 ± 7.3 55±5.2 107±11.1 52 ± 4.8 741 ± 52.8 122 _ 10.9 115 ± 8.5
m
_
41 55 65 59
± ± ± ±
i
60 63 71 65
_ 3.4 ± 4.9 ± 5.5 ±4.8
57 ± 65 ± 77 ± 67±
4.1 5.2 5.8 5.1
* P ~ 0.01 compared with corresponding controls - < 10 pmol/g.
Postnatal days 0 and 7 At the day o f birth, i m m u n o r e a c t i v e M E levels a n d M E r e c e p t o r b i n d i n g in 10 a n d 9 n u c l e i respectively increased rapidly w h e r e a s h a r p 2 - 3 fold i n c r e a s e w a s n o t i c e d . Both l e v e l s a b r u p t l y a p p e a r e d in 65 o u t o f 73 n u c l e i in w h i c h b o t h v alues w e r e n o t d e t e c t a b l e d u r i n g t h e p r e n a t a l p e r i o d ( T a b l e s III a n d IV). B o t h l e v e l s i n d i c a t e d the a d u l t p a t t e r n a n d w e r e p a r t i c u l a r l y h i g h in the n. t r a c t u s s o l it a r i i ( c a u d a l part), n. p a r a b r a chialis, n. a c c u m b e n s s e p t i a n d g r o u p o f t h e stria t e r m i n a l n u c l e i . B e t w e e n p o s t n a t a l d a y s 0 a n d 7,
most nuclei on the postnatal day 7 were higher than those at 4 and 20 weeks of age 32. During the neonatal period, SHR had lower ME levels in the tractus spinalis nervi trigemini (Vts) and ME receptor binding in the n. tractus spinalis nervi trigemini (Vnts) than those of WKY (Fig. 1). The difference persisted in young SHR at the age of 4 weeks and disappeared in adult SHR at age of 20 weeks32. A ntinociceptive threshold T a b l e V s h o w s t h e d i f f e r e n c e in a n t i n o c i c e p -
both M E levels and r e c e p t o r b i n d i n g increased
tive s e n s i t i v i t y o f n e o n a t a l , y o u n g a n d
by 5-20% a n d 2 0 - 4 0 % , r e s p e c t i v e l y . T h e a b s o lute v a l u e s o f M E l e v e l s a n d r e c e p t o r b i n d i n g in
SHR a n d W K Y . S l i g h t b u t s i g n i f i c a n t d i f f e r -
adult
e n c e s c o u l d b e d e m o n s t r a t e d in all a n i m a l s . In
28 Tractus s P l n o l l s nervl t r l o e m l n l
400
I
blrtn
~~o t ~ ~ ~ ~EL 0 WKY • SHR
~
tained in y o u n g S H R (4 weeks old) by the hotplate method. However, adult S H R (20 weeks old) showed a relative antinociceptive effect when c o m p a r e d with normotensive W K Y o f the same age in both tests (Table V).
MERB
200 "
DISCUSSION
P~O,05
100
100 '
a
001"4
D-~
LI
I
l
4
20
blrth~
|1
-I
Nucleus troctus sotnolls
0
i
I
1
•
x.
20
nervl trlg~lnl
400
~E IL I
Evidence presented in this p a p e r indicates m a r k e d heterogeneity o f silver grain distribution in various brain areas and sharp boundaries o f grain densities in certain areas, which indicate that observed localizations o f both M E levels and M E receptor binding are not d e t e r m i n e d by diffusions but represent the distribution o f both levels. F u r t h e r m o r e , the possibility that [3H]decomposed product may bind to non-selective sites can be excluded, since regional selectively o f grain and extent o f grain densities are in accordance with those o f M E levels. Non-specific binding a m o u n t s to a b o u t 3% o f the total binding in most nuclei. Our results indicated that the regional distribution o f i m m u n o r e a c t i v e M E and its receptor binding could be detected respectively in 10 and 4 o f 83 cerebral nuclei on the e m b r y o n i c day 14, and also 10 and 9 nuclei on the e m b r y o n i c day 18, 3 days before birth. This indicates that the prenatal d e v e l o p m e n t o f cerebral M E neurons is limited to some nuclei and that M E receptor systems in the prenatal period develop later than ME i m m u n o r e a c t i v e cells. During the prenatal period of d e v e l o p m e n t in these limited nuclei, the levels o f M E and receptor binding increased at a higher rate than those in the early postnatal period. T h e finding is consistent with the pre-
MERB birth
g ~.200 100
0 01--~ LI
i
-
0 i
M,
1
q
20
Time In
-I
weeksafterDtrtn
0
I
tl
20
Fig. 1. Development of methionine-enkephalin-like immunoreactivity (MELI) and [3H]methionine-enkephalin receptor binding (MERB) in the Vts and Vnts of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Values are expressed as means --. S.E.M. for 8 animals per group. Data for both animals of 4 and 20 weeks after birth are those as described previously32.
both animal groups o f 1 and 4 weeks o f age, antinociceptive thresholds o f S H R were slightly but significantly (P < 0.05) lower in the tail-flick test than those o f W K Y . Similar results were obTABLE V
A ntinociceptive thresholds in male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats ( W K Y)
The data represent means ± S.E.M. for the number of animals shown in parenthesis. A ge weeks
n
1 4 20
9 15 16
* P < 0.05.
** P < 0.01.
Tailflick (latency, s)
Hot-plate (latency, s)
WK Y
SHR
% difference
WK Y
SHR
% difference
1.48 -4-0.054 3.02 ± 0.205 3.03 ± 0.219
1.33 ± 0.0372* 2.53 ± 0.114" 5.29 ± 0.728**
- 11 -16 +75
n.d. 11.8 ± 0.972 9.84 ± 0.974
n.d. 9.04 ± 0.755* 17.3 +__1.93"*
23 +76
29 vious reportss,9 that there is an initial appearance of [3H]opiate antagonist binding from embryonic day 15 through birth followed by a slower increase to adult levels. The localization of both levels in most nuclei which abruptly appeared on the day of birth indicated the adult pattern. There was no rank order correlation between both levels presented in the prenatal and postnatal periods. This process of the delayed detection of immunoreactive ME is quite in parallel to that of immunoreactive LE in rat embryonic brain regions in which endorphin maturation is much faster2. The abrupt appearance of ME and receptor binding of neonatal rats coincides closely with an abrupt increase in the pain thresholds in dams 1-2 days before parturition. This antinociceptive action is present only during pregnancy and is abolished by naltrexone administration ~4. The full-term human placenta contains immunoreactive ME and fl-endorphin of which the local release may regulate sensory transmission from the uterus and vaginal tract during childbirth41. During the embryonic and neonatal periods of 4 developmental time-points we studied, ME receptor binding in various nuclei was the highest on the postnatal day 7. The results are consistent with the previous findings of a rapid increase from birth to weaning of [3H]ME receptor39,50,57 and [3H]opiate agonist and antagonist receptors8.9,38,57. Such early postnatal development of ME neurons may be be responsible for neonatal withdrawal symptoms in infants of addicted mothers5°. During the period of the rat neonatal days 0 and 7, ME levels in the Vts and ME receptor binding in the Vnts in the medulla oblongata were markedly lower in SHR than in WKY. The difference persisted in young SHR at age of 4 weeks but not in adult SHR at age of 20 weeks32. The Vnts caudal and rostral 5 known to receive afferent terminals from the facial and oral areas, to send both ascending fibers to the ventral thalamus 13.44 and descending fibers to the spinal cord 7,42,49, thus modulating sensory impulses in the spinal dorsal horn. Particularly in the rodent, the Vnts receives afferent inputs from the mystacial vibrissae ~L'2, which are the important sniff-
ing-linked tactile sensory organs in the rat 52. Since nociceptive thresholds of neonatal SHR determined presently by two methods were lower than those of neonatal WKY, noxious sensory perception of SHR neonates might be greater than that of WKY neonates. Although decreased reactivity to noxious stimuli has been reported in SHR 53 and in rats with raised blood pressure J°,55.56, these animals are adult or young but not neonate. More reckless and hyper-reactive behavior of neonatal SHR apparently heightened pain sensitivity in the present study. However, adult SHR exhibited an antinociceptire effect when compared with normotensive adult SHR, in agreement with rats with raised blood pressure 1°.55.56. Thus, the decreased ME and receptor binding in the Vts and Vnts of SHR neonates probably disinhibit afferent noxious as well as non-noxious sensory information. Such a genetic deficiency might then produce effects on the proper development of the CNS system, similar to the deleterious effects of sensory deprivation on the ontogenetic development of the visual system. In young SHR, prolonged deprivation of such environmental stimuli as social contacts and confrontations 15 and light28 attenuates and delays the rise of the mean blood pressure as paralleled by less pronounced cardiovascular structural changes. Furthermore, electrical stimulation of the Vts and Vnts area at the level of the obex is known to produce hypotension and bradycardia in decerebrate or anesthetized rabbits, which is termed the trigeminal depressor response (TDR) 26. Its hemodynamic pattern is similar to aortic depressor reflex25. In view of rich presence of ME immunoreactivity and receptor in the area, the participation of ME neuronal activity with the TDR is highly probable. In SHR neonates, the average arterial blood pressure is very small but already significantly higher than that in WKY 6,17. In SHR, the neonatal deficiency of ME neurons in the Vts and Vnts was also confirmed to precede with the following expansive abnormality in 4 weeks of age: the decreased ME levels and receptor binding in the n.reticularis lateralis, n. arcuatus and n. corporis mammillaris, and the increased ME levels and receptor binding in
30 the n. dorsalis nervi vagi, n. amygdaloideus medialis and centralis, and group of the stria terminal nuclei 32'33. The ME neuronal activation in these latter nuclei is consonant with the previous finding of the enhanced glucose utilization rate in the same nuclei ~7. In young SHR, there is preganglionic-dependent activation of sympathetic nervous system 35,36, and central hyperreactivity accompanied by a greater sympathetic activation 16,36.These results suggest that there is an ontogenic correlation between enhanced sensory perception and sympathetic activation. In conclusion, at the day of birth ME neurons
abruptly appeared in most cerebral nuclei except for several nuclei already present in the prenatal period. During the perinatal period of SHR, ME levels in the Vts and ME receptor binding in the Vnts were markedly lower in SHR than WKY neonates. The neonatal ME neuronal deficiency in these areas leading to further ME abnormality in the hypothalamus, strio-amygdaloid complex and n. dorsalis nervi vagi 32 may be prerequisite for the development of sympathetic activation and hyper-reactivity in young SHR.
REFERENCES
12 Erzurumlu, R. S. and Killackey, H. R., Order in the developing rat trigeminal nerve, Develop. Brain Res., 3 (1982) 30:%310. 13 Fukushima, T. and Kerr, F. W. L., Organization of trigemino-thalamic tracts and other thalamic afferent systems of the brainstem in the rat: presence of gelatinosa neurons with thalamic connections, J. comp. Neurol., 183 (1979) 169- 184. 14 Gintzler, A. R., Endorphin-mediated increases in pain threshold during pregnancy, Science, 210 (1980) 193 195. 15 Hallb~ick, M., Consequence of social isolation on blood pressure, cardiovascular reactivity and design in spontaneously hypertensive rats, A cta physiol, stand., 93 (1975) 455-465. 16 Hallb/ick, M. and Folkow, B., Cardiovascular responses to acute mental 'stress' in spontaneously hypertensive rats, A ctaphvsiol, scand., 90 ( t 974) 684-698. 17 Hayashi, T. and Nakamura, K.. Activation of cerebral neuronal activity in spontaneously hypertensive rats as devaonstrated by the 14C-deoxyglucose method, NaunvnSchmiedeberg's Arch. Pharmacol., 316 ( 1981 ) 331- 339. 18 Heybach, J. P. and Danellis, J. V., The effect of pituitaryadrenal function in the modulation of pain sensitivity in the rat, J. Physiol. (Lond.), 283 (1978) 331- 340. 19 H6kfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in the central neurons system, Neurosci. Lett., 5 (1977) 25-31. 20 H6kfelt, T., Terenius, L., Kuypers, H. G. J. M. and Dann, O., Evidence for enkephalin immunoreactive neurons in the medulla oblongata projecting to the spinal cord, Neurosci. Lett., 14 (1979) 55- 60. 21 Hong, J. S., Yang, H.-Y. T., Eratta, W. and Costa, E., Determination ofmethionine enkephalin in discrete regions of rat brain, Brain Research, 134 (1977) 383- 386. 22 Hughes, J., Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine, Brain Research, 88 (1975) 295- 308. 23 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 (1976) 577~ 579.
1 Bardo, M. T., Bhatnagar, R. K. and Gebhart, G. F.. Opiate receptor ontogeny and morphine-induced effects: influence of chronic footshock stress in preweanling rats, Develop. Brain Res., 1 ( 1981 ) 487-495. 2 Bayon, A., Shoemaker, W., Bloom, F. E., Mauss, A. and Guillemin, R., Perinatal development of the endorphinand leucine enkephalin-containing systems in the rat brain, Brain Research, 179 (1979) 93-101. 3 Bellet, M., Elghozi, J. L., Meyer, P., Pernolett, M. G. and Schmitt, H., Central cardiovascular effects of narcotic analgesics and enkephalins in rats, Brit. J. Pharmacol., 71 (1980) 365-369. 4 Bolme, P., Fuxe, K., Agnati, L. F., Bradley, R. and Smythies, J., Cardiovascular effects of morphine and opioid peptides following intracisternal administration in chloralose-anesthetized rats, Europ. J. Pharmacol., 48 (1978) 319--324. 5 Broton, J. G. and Rosenfeld, J. P., Rostral trigminal projections signal perioral facial pain, Brain Research, 243 (1982) 395-400. 6 Bruno, L., Azar, S. and Weller, D., Absence ofa pre-hypertensive stage in post-natal Kyoto hypertensive rats, Jap. HeartJ., 20 Suppl. I (1979) 90-92. 7 Burton, H., Craig, A. D., Poulos, D. A. and Molt, J. T., Efferent projections from temperature sensitive recording loci within the marginal zone of the nucleus candalis of the spinal trigeminal complex in the cat, J. comp. Neurol., 183 (1979) 753-778. 8 Clendeninn, N. J., Petraitis, M. and Simon, E. J., Ontological development of opiate receptors in rodent brain, Brain Research, 118 (1976) 157-160. 9 Coyle, J. T. and Pert, C. B., Ontogenetic development of [3H]naloxone binding in rat brain, Neuropharmacol., 15 (1976) 555- 560. l0 Dworkin, B. R., Filewich, R. J., Miller, N. E., Craigmyle, N. and Pickering, T. G., Baroreceptor activation reduces reactivity to noxious stimulation: implications for hypertension, Science, 205 (1979) 1299-1301. 11 Erzurumlu, R. S., Bates, C. A. and Killackey, H. P., Differential organization of the thalamic projection cells in the brain stem trigeminal complex of the rat, Brain Research, 198 (1980) 427-433.
31 24 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, R. E. Krieger, New York, 1970. 25 Kumada, M., Dampney, R. A. L., Whitnall, M. H. and Reis, D. J., Hemodynamic similarities between the trigeminal and aortic vasodepressor responses, Amer. J. Physiol., 234 (1978) H6%H73. 26 Kumada, M., Dampney, R. A. L. and Reis, D. J., The trigeminal depressor response: a novel vasodepressor response originating from the trigeminal system, Brain Research, 119 (1977) 305-326. 27 Kuraishi, Y., Sugimoto, M., Hirota, N. and Takagi, H., Lack of effect of morphine and noxious stimuli on the Met-enkephalin content in the spinal dorsal horn and nucleus reticularis gigantocellularis of the rat, Life Sci., 30 (1981) 2071-2077. 28 Lais, L. T., Bhatnagar, R. A. and Brody, M. J., Inhibition by dark adaptation of the progress of hypertension in spontaneously hypertensive rat (SHR), Circulat. Res., 34 and 35, Suppl. I (1974) 155-160. 29 Laubie, M. and Schmitt, H., Indication for central vagal endorphinergic control of heart rate in dogs, Europ. J. Pharmacol., 71 (1981)401-409. 30 Laubie, M., Schmitt, H., Vincent, M. and Resmond, G., Central cardiovascular effects of morphinomimetic peptides in dogs, Europ. J. Pharmacol., 46 (1977) 67-71. 31 Lord, J. A. H., Waterfield, A. A., Hughes, J. and Kosterlitz, H. W., Endogenous opioid peptide: multiple agonists and receptors, Nature (Lond.), 267 (1977) 495-499. 32 Nakamura, K. and Hayashi, T., Methionine enkephalinergic neuronal activity in cerebral nuclei of spontaneously hypertensive rats, Hypertension, 4 (1982) 662669. 33 Nakamura, K. and Hayashi, T., Altered methionine enkephalin immunoreactivity in cerebral nuclei of spontaneously hypertensive rats, Clin. exp. Hyp., A4 (1982) 297-301. 34 Nakamura, K., Kuntzman, R., Maggio, A. C., Augulis, V. and Conney, A. H., Influence of 6-hydroxydopamine on the effect of morphine on the tail-flick latency, Psychopharmacol., 31 (1973) 177-189. 35 Nakamura, K. and Nakamura, K., Selective activation of sympathetic ganglia in young, spontaneously hypertensive rats, Nature (Lond.), 266 (1977) 265-266. 36 Nakamura, K. and Nakamura, K., Role of brainstem and spinal noradrenergic and adrenergic neurons in the development and maintenance of hypertension in spontaneously hypertensive rats, Naunyn-Schmiedeberg's Arch. Pharmacol., 305 (1978) 127-133. 37 Palkovits, M. and Jacobowitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain - II.Hindbrain (mesencephalon, rhombencephalon), J. comp. Neurol., 157 (1974) 2% 42. 38 Pasternak, G. W., Zhang, A. and Tecott, L., Developmental differences between high and low affinity opiate binding sites: their relationship to analgesia and respiratory depression, Life Sci., 27 (1980) 1185-1190. 39 Patey, G., Baume, S., Gros, C. and Schwartz, J., Ontogenesis of enkephalinergic systems in rat brain: postnatal changes in enkephalin levels, receptors and degrading enzyme activities, Life Sci., 27 (1980) 245 252. 40 Petty, M. A. and Reid, J. L., Opiate analogs, substance P and baroreceptor reflexes in the rabbit, Hypertension, 3 Suppl. I (1981) 142- 147. 41 Rama Sastry, B. V., Barnwell, S. L., Tayeb, O. S., Janson,
V. E. and Owens, L. K., Occurrence of methionine enkephalin in human placental villus, Biochem. Pharmacol., 29 (1980) 475-478. 42 Ruggiero, D. A., Ross, C. A. and Reis, D. J., Projections from the spinal trigemlnal nucleus to the entire length of the spinal cord in the rat, Brain Research, 225 (1981) 225233. 43 Schaz, K., Stock, G., Simon, W., Schl6r, K.-H., Unger, T., Rockhold, R. and Ganten, D., Enkephalin effects on blood pressure, heart rate, and baroreceptor reflex, Hypertension, 2 (1980) 395- 407. 44 Shigenaga, Y., Takabatake, M., Sugimoto, T. and Sakai, A., Neurons in marginal layer of trigeminal nucleus caudalis projecting ventrobasal complex (VB) and posterior nuclear groups (PO) demonstrated by retrograde labeling with horseradish peroxidase, Brain Research, 166 (1979)391 396. 45 Simantov, R., Kuhar, M. J., Uhl, G. R. and Snyder, S. H., Opioid peptide enkephalin: immunohistochemical mapping in rat central neurons system, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 2167-2171. 46 Simantov, R. and Snyder, S. H., Morphine-like factors in human brain: structure elucidation and interactions with the opiate receptor, Proc. nat. Acad Sci. U.S.A., 73 (1976) 2515-2519. 47 Smith, T. W., Hughes, J., Kosterlitz, H. W. and Sosa, R. P., Enkephalins: isolation, distribution and function. In H. W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides, North-Holland, Amsterdam, 1977, pp. 5~62. 48 Stengaard-Petersen, K. and Larsson, L.-I., Comparative immunocytochemical localization of putative opioid ligands in the central neurons system, Histochemistry, 73 (1981)8%114. 49 Tohyama, M., Sakai, K., Salvert, D., Touret, M. and Jouvet, M., Spinal projections from the lower brain stem in the cat as demonstrated by the horseradish peroxidase technique. I. Origins of the reticulospinal tracts and their funicular trajectories, Brain Research, 173 (1979) 383403. 50 Tsang, D. and Ng, S.C., Effect of antenatal exposure to opiates on the development of opiate receptors in rat brain, Brain Research, 188 (1980) 199-206. 51 Uhl, G. R., Goodman, R. R., Kuhar, M. J., Childers, S. R. and Snyder, S. H., Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Research, 166 (1979) 75-94. 52 Welker, W. I., Aalysis of sniffing of the albino rat, Behaviour, 22 (1964) 223-244. 53 Wendel, O. T. and Bennett, B., The occurrence of analgesia in an animal model of hypertension, Life Sci., 29 (1981) 51~521. 54 Yang, H.-Y., Hong, J. S. and Costa, E., Regional distribution of Leu- and Met-enkephalin in rat brain, Neuropharmacol., 16 (1977) 303-307. 55 Zamir, N. and Segal, M., Hypertension-induced analgesia: changes in pain sensitivity in experimental hypertensive rats, Brain Research, 160 (1979) 170- 173. 56 Zamir, N. R., Simantov, R. and Segal, M., Pain sensitivity and opioid activity in genetically and experimentally hypertensive rats, Brain Research, 184 (1980) 299- 310. 57 Zhang, A.-Z. and Pasternak, G. W., Ontogeny of opioid pharmacology and receptors: high and low affinity site differences, Europ. J. PharmacoL, 73 (1981) 29- 40.
21
Development of Cerebral Methionine-Enkephalinergic Neurons in Rats: Some Difference in Wistar-Kyoto Rats and Spontaneously Hypertensive Rats KEIJI NAKAMURA and TETSUO HAYASHI
Department of Pharmacology, Nippon Roche Research Center, Kamakura - 247 (Japan) (Accepted December 7th, 1982)
Key words: methionine-enkephalin- opioid neurons - cerebral nuclei - development - spontaneously hypertensive rats
Development of methionine-enkephalin(ME) and ME receptor binding in the embryonic and neonatal rat cerebral nuclei was defined quantitatively by immunocytofluorescentand microautoradiographic methods. On embryonic days 14 and 18, ME was localized in I0 of 83 nuclei particularly in the n. amygdaloideus centralis, n. periventricularis, n. supraopticus, n. interpeduncularis, n. suprachiasmaticus, n. arcuatus and n. ambiguus. ME receptor binding was distributed in the former 4 nuclei on embryonic day 14 and additional 5 nuclei on embryonic day 18. At the day of birth both levels markedly increased in these nuclei (2-3 times) and abruptly emerged in 65 out of 73 nuclei in which ME neurons were not detectable in embryonic nuclei. The abrupt appearance in most nuclei at birth probably regulates nociceptive and non-nociceptivestimuli at birth and in the neonatal period. Both levels attained their maximum at the postnatal day 7 and gradually declined thereafter. Among the perinatal period examined, ME immunoreactivity in the tractus spinalis nervi trigemini and ME receptor binding in the n. tractus spinalis nervi trigemini were markedly lower in both SHR neonates than in corresponding Wistar-Kyoto rats. Lowered levels in both areas of neonatal SHR may be involved in central hyperreactivity and preganglionic sympathetic activation of young SHR. INTRODUCTION
The enkephalin pentapeptides 22,23,46 have a widespread distribution in the brain. The binding of radiolabeled enkephalins directly to brain membranes is described 31.57. In the adult rat brain, methionine-enkephalin (ME) levels are found to be about three47 or severaP 4 times higher than leucine-enkephalin (LE) levels. In the adult rat brain, there are regional mappings for enkephalins with the antisera to both ME and LE45,48m and with the antisera to ME with some cross-reactivity to LE 19-21. There are several reports describing ontological alterations of immunoreactive enkephalinsz,39 and synthetic [3H]opiate agonist and antagonist bindings in the rat brain regions l,z,s,9.5° and the rat whole brain homogenates9,38,57. However, until recently, selective antisera to ME for quantitative analysis of cerebral nuclei and also ME-receptor binding in nuclei had not been available 32. Thus, 0006-8993/ 83 / $03.00 © 1983 Elsevier Science Publishers B.V.
developmental alteration of immunoreactive and receptor binding levels in various cerebral nuclei might be difficult to detect. Recent functional studies suggest that endogenous enkephalins are involved in cerebral cardiovascular, control, since naloxone-reversal alteration of blood pressure and heart rate is produced by the intracisternal or intracerebral administration of metabolically stable synthetic MEs 3,4,30,40,43and putative enkephalin releasers29. In some cerebral nuclei of young and adult spontaneously hypertensive rats (SHR), there are alterations of immunoreactive ME and receptor binding levels as compared to those of normotensive Wistar- Kyoto rats (WKY) 32.33. In this report we now present evidence that ME and ME receptor binding levels are distributed in the limited nuclei of prenatal rat brains and abruptly appear in most of cerebral nuclei in the day of birth. Although there is no difference in both levels between SHR and WKY dur-
ing the prenatal period, there are lowered ME levels in the tractus spinalis nervi trigemini (Vts) and ME receptor binding in the n. tractus spinalis nervi trigemini (Vnts) of SHR at the early postnatal period. MATERIAL AND METHODS
The experiments were performed on 4 paired groups of male S H R and age-matched male normotensive W K Y raised in our animal facilities. The animals used were embryonic days 14 and 18 as estimated by vaginal plug formation, the day of birth and one week after birth. In 83 different cerebral nuclei, tracts and areas, both ME contents and ME receptor binding were measured quantitatively by autoradiograms prepared by the immunohistofluorescence antibody and by [3H]ME respectively, as described previously32'33. In brief, for measuring immunoreactive ME levels of the cerebral nuclei, the animals were sacrificed by intracardiac perfusion of icecold 4% paraformaldehyde in a buffered solution. From each frozen brain, coronal sections (25 ~m) were cut serially and processed as described previously 32-3~-45. They were then mounted on glass cover-slips and brought to reaction for 30 min at 37°C with 5-fold diluted anti-ME rabbits sera. The antiserum, a gift of Drs. Takagi and Kuraishi (Kyoto), was not cross-reactive with/~-endorphin (less than 0.04%) and with LE (0.008%)27. The treated sections were subsequently incubated for 15 min at 37 °C with fluorescence-conjugated goat antibody (20-fold diluted) against rabbit IgG (Difco). For standards, we used 8% gelatine sections containing 8 different concentrations ( ~ 1280 pmol/g) of ME. The silver granule density of the fluorescence micrographs obtained by exposure to Kodak plus X was measured with a microdensitometer (Gamma Scientific) in cerebral areas of 50 ~m diameter. For measuring MERB, the animals were sacrificed by cardiac perfusion of ice-cold 0.1% formaldehyde in a buffered solution (pH 7.4). From each frozen brain, cryostat brain sections (5 ~m) were mounted on microscope slides and
incubated for 60 min at 40"C with 4 nM of pH]ME (spec. act. of 40-60 Ci/mmol, New England Nuclear) as described previouslf :, For the non-specific binding, unlabeled ME (4~M) was further added to the incubated medium on the mounted tissues. Non-specific binding which was defined as the binding in the presence of unlabeled ME amounted to about 3% of the total binding in most nuclei. For reference standards, we used 8% gelatine slices (5 /,m) containing 9 different [3H]ME concentrations (0 1280 pmol/g). After cover-slips were dipped in a solution of Kodak NTB-2 photographic emulsion (2-fold diluted), the emulsion-coated cover slips were dried and glued onto the slides. Cover-slip and slide were clamped tightly together with a clip and kept in the light-proof condition until the exposure was complete (8-10 weeks). The autoradiographic image on the cover-slip was then developed, and the silver granule density (200 >m diameter) was measured with the microdensitometer. Brain anatomy and terminology were as described by K6nig and Klippe124 and Palkovits and Jacobowitz 3v. Antinociceptive threshold was determined by a minor modification of the hot-plate test for rats as previously described ~.53. Tests were performed in a plexiglass chamber with dimensions of 25 × 25 x 25 cm. The floor of the chamber was constantly heated at 55 m 0.3 °C by circulation of warm water. The threshold was measured by the latency to lick the forepaw or j u m p from the floor. Each animal was tested 3 times at 10 min intervals, and an average response of the last two times was calculated. This procedure eliminated any novelty influence of handling or heat exposure on test results. To avoid abdominal burning, the hot-plate test for neonates was not performed. In another test pain threshold was determined by the rat tail flick method as previously described 34. Heat from an infra-red lamp was focused on the tip of the tail of animals. The latent time to remove the tail was measured 3 times at 10 min intervals and the results of the last twice averaged. Aortic blood pressure was measured in some dams without anesthesia and restraint with a pressure transducer (Nihon Koden, MPU0.5) through a ca-
23 TABLE I
Cerebral nuclei with the presence of methionine-enkephalin-like immunoreactivity in the embryo and neonate of normotensive Wistar- Kyoto rats (WKY) and spontaneously hypertensive rats (SH R) Data ( p m o l / g ) are m e a n s __. S.E.M. for 8 animals per each group.
CerebraI n uclei
R ats
Embryo nic days 14
N.ambiguus N. interpeduncularis N. reticularis lateralis N. supraopticus N. suprachiasmaticus N. periventricularis N.arcuatus N. amygdaloideus lateralis N. amygdaloideus centralis N. amygdaloideus medialis
WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR
88 89 163 151 54 55 211 209 241 255 296 266 211 225 33 36 277 276 58 65
Neon ataI days 18
+ 5.9 ___ 6.4 ± 10.7 ± 9.5 __. 3.7 __. 4.4 ± 15.2 __. 13.4 ± 16.3 __. 20.7 +-- 22.7 + 17.4 __. 16.3 ± 13.5 --. 2.2 ± 1.9 __. 18.5 ± 17.3 +-. 3.9 +_. 4.1
123 120 236 219 62 63 258 266 318 309 336 329 274 241 42 42 334 345 82 81
0 __. 8.4 __. 8.9 ± 22.4 ± 15.6 ± 4.5 __. 4.7 __. 16.4 __. 15.9 __. 22.8 +_. 21.6 ± 22.9 + 21.7 __. 21.5 ± 20.8 __. 3.4 ± 2.5 __. 22.7 ___ 26.3 __. 3.1 __. 6.2
381 368 627 620 95 81 671 690 751 781 966 918 677 709 113 109 849 852 142 152
7 __. 26.4 __. 31.5 ± 48.2 __. 40.1 __. 7.6 ± 6.3 __. 49.2 __. 58.4 ± 57.4 __. 68.3 ___ 65.9 ± 76.5 ± 49.3 ± 51.7 __. 8.7 ± 7.8 __. 67.2 ___ 63.1 ± 10.5 __. 9.7
409 387 684 680 105 97 741 732 849 819 1,015 1,059 770 785 127 135 916 935 172 161
__. 27.6 __. 33.9 __. 51.7 ± 51.9 __. 8.6 ± 6.5 ± 57.3 ± 55.1 ± 67.4 ± 75.9 __. 84.7 __. 72.6 ± 60.5 __. 58.8 ± 7.9 __. 8.5 __. 62.7 ± 71.2 ___ 10.5 __. 11.3
T A B L E II
Cerebral nuclei with the presence of methionine-enkephalin receptor binding in the embryo and neonate of normotensive WistarKyoto rats (WK Y) and spontaneously hypertensive rats (SHR) Data ( p m o l / g ) are m e a n s ± S.E.M. for 5 animals per each group.
Cerebral nuclei
N.ambiguus N. interpeduncularis N. reticularis lateralis N.supraopticus N. suprachiasmaticus N. periventricularis N. arcuatus N. amygdaloideus centralis N. amygdaloideus medialis
Group
WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR
Embryonic days
Neonatal days
14
18
~10 ~ 10 86 __. 9.3 91 ± 8.7 ~ 10 < 10 123 ± 16.4 182 ± 14.7 < 10 ~10 186 ± 20.5 167 __. 17.8 < 10 <10 210 ± 19.6 221 ___ 21.5 ~ 10 ~10
51 55 148 163 41 46 165 182 224 241 247 236 186 167 242 266 53 48
0 ± 4.8 __. 6.3 __. 16.5 ± 18.2 ± 4.3 ± 5.2 ± 19.5 ± 19.3 __. 26.3 __. 25.8 ± 25.5 ± 24.8 __. 20.4 __. 18.3 ± 26.3 ± 28.5 ± 5.6 __. 5.1
246 263 413 427 68 59 481 446 496 525 601 638 434 411 574 583 96 89
7 __. 21.3 ___ 22.8 __. 39.6 ± 41.4 ± 6.3 ± 6.7 __. 40.9 ± 38.1 ± 41.5 ± 43.9 ± 57.4 ± 59.5 ± 40.1 ___ 32.7 ± 46.3 ± 52.6 ± 8.4 ± 9.1
315 326 502 483 81 85 549 568 589 558 675 681 478 485 669 643 103 ! 12
__. 28.1 __. 25.0 __- 42.6 ± 41.8 __.8.3 __. 7.5 __. 50.7 __. 67.5 ± 50.6 __. 48.2 ___ 61.5 __. 64.4 + 42.3 ± 45.6 ± 58.4 ± 62.8 __. 9.6 __. 9.3
24 T A B L E II1
Methionine-enkephalin-like immunoreactivity in cerebral n uclei of neonates of normotensive Wistar-- Kvoto rats ( WK Y) and spontaneous!v hypertensive rats (SHR) in which M E immunoreactivity was not detected in embryonic brains Data ( p m o l / g ) are m e a n s 4- S.E for 8 animals.
Structure
Postpartum Day 0
%change
WK Y (8) Rhombencephalon and mesencephalon N. intercalatus N.commissuralis Areapostrema N. tractus solitarii (caudal) N . o r i g i n i s n. hypoglossi N . d o r s a l i s n. vagi N. olivaris inferior N. tractus spinalis n. trigemini T r a c t u s s p i n a l i s n . trigemini N. p a r a b r a c h i a l i s dorsalis N. p a r a b r a c h i a l i s v e n t r a l i s S u b s t a n t i a n i g r u s pars c o m p a c t a S u b s t a n t i a grisea centralis
107 418 179 870 311 318 266 344 1086 1044
4- 8.4 4- 28.7 4- 13.6 4- 66.3 4- 25.4 ___ 26.5 4444-
20.5 27.6 82.5 96.3
SHR (8) 111 406 185 841 301 308 249 231 1044 1014
4- 9.9 4- 34.6 4- 16.4 4- 64.7 4- 25.3 ___ 22.6 4444-
21.7 16.5" 76.1 76.6
-33
Day 7
% change
WK Y (8)
SHR (8)
125 457 221 908 340 362
128 443 219 891 351 358
293 395 1120 1106
4- 10.8 4- 35.7 4- 15.4 4- 76.5 ___ 26.5 4- 28.1
444444-
10.7 33.2 17,5 83.6 27.7 26.4
4- 22.3 286 4- 23.5 4- 27.4 263 4- 24,2* 4- 96.9 1 1 0 2 4 - 9 1 . 4 ___ 104.2 1078 4- 83.5
506 4- 42.3
491 ___ 33.6
532 4- 46.4
510 4- 38.7
Thalamus and epithalamus N. p a r a m e d i a n u s N. c e n t r o m e d i a n u s N. d o r s o m e d i a l i s N. v e n t r o m e d i a l i s N. h a b e n u l a e m e d i a l i s N. h a b e n u l a e lateralis
105 85 89 81 142 155
444444-
7.5 6.4 7.1 5.7 10.5 8.7
98 87 92 84 151 143
444444-
7.3 6.1 6.8 6.3 8.5 11.7
115 98 103 94 150 156
107 101 97 90 156 150
4- 7.3 ___ 6.3 ___ 7.6 4- 7.1 ___ 10.4 --- 11.2
F o r m a t i o reticularis N. r a p h e o b s c u r u s N. r a p h e p a l l i d u s N.raphemagnus N. t e g m e n t i d o r s a l i s N. t e g m e n t i ventralis N. l i n e a r i s m e s e n c e p h a l i N. reticularis centralis N. reticularis gigantocellularis N. reticularis paragigantocellularis N. r u b e r m e s e n c e p h a l i N. reticularis parvocellularis N. reticularis m e d i a l i s N. reticularis dorsalis S u b s t a n t i a reticularis m e s e n c e p h a l i Z o n a incerta N. intralaminalis N. reticularis t h a l a m i
237 215 284 963 563 236 57 59 53 144 120 88 51 76 74 53 -
4- 21.3 4- 15.4 4- 18.5 4- 65.4 4- 39.5 4- 17.5 4- 4.4 4- 4.7 4- 4,1 4- 10.5 4- 8.9 4- 6.2 __. 4.3 ___ 6.5 ___ 6.2 4- 4.1
226 218 276 1016 533 248 59 55 78 149 117 95 50 79 78 51 -
4444444444444444-
16.3 17.8 21.5 73.9 38.7 16.9 5.2 4.6 4.1 10.7 8.5 7.1 4,1 7.2 5.3 4. I
266 236 324 1084 634 268 64 64 60 166 133 95 59 91 86 60
21.2 16.4 21.3 72.5 41.7 15.8 4.2 5.1 5.1 11.2 8.4 7.0 3.7 7.1 6.3 5.6
243 237 318 1088 635 281 65 65 62 158 136 97 63 87 83 62
4- 18.7 4- 18.5 4- 26.4 __. 75.2 4- 44.2 ___ 17.7 4- 5.3 --- 4.1 + 4.2 ___ 11.8 4- 9.1 4- 7.5 --- 4.3 4- 7.2 --- 4.9 4- 4.2
Hypothalamus N. p o s t e r i o r h y p o laalami N. corporis m a m m i l l a r i s medialis N. corporis m a m m i l l a r i s lateralis N. v e n t r o m e d i a l i s N. d o r s o m e d i a l i s Area lateralis h y p o t h a l a m i Area anterior h y p o t h a l a m i N. paraventricularis Median eminence lnfundibulum
108 61 56 105 99 188 176 585 65 -
__. 8.4 4- 4.7 --- 3.0 4- 9.5 4- 7.8 4- 14.3 4- 14.2 4- 44.2 4- 5.1
113 58 54 102 105 181 181 565 60 -
4- 8.5 4- 4.4 __. 3.7 __. 9.1 __. 7.6 __. 14.9 4- 15.2 4- 43.6 4- 4.2
120 75 64 116 111 205 208 647 74
__. 8.6 4- 5.9 __. 3.8 __- 8.7 __. 6.5 4- 15.8 4- 16.4 ___ 42.3 __. 4.2
119 77 63 111 118 217 215 668 75
__. 8.5 __. 5.8 __. 4.3 _4- 10.5 4- 8.7 4- 13.1 ___ 18.2 4- 40.2 4- 5.3
4- 8.4 ___ 7.6 ___ 7.6 4- 6.5 ___ 11.3 4- 13.4
4444444444444444-
~--33
25 TABLE III
(Continued)
Structure
Postpartum 56change
Day 0
WKY (8) Fasciculus medialis telencephali (caudal) Fasciculus medialis telencephali (rostral) Fasciculuslongtidunalisdorsalis Forel H Telencephalon N. amygdaloideus intercalatus N. amygdaloideus basalis pars lateralis N. amygdaloideus basalis pars medialis N. araygdaloideus corticalis Area amygdaloidea anterior N. tractus olfactorius lateralis N. proprius striae terminalis N. interstitialis striae terminalis dorsalis N. interstitialis striae terminalis ventralis N. interstitialis striae terminalis medullaris N. dorsalis septi N. medialis septi N. lateralis septi N. intermedius septi N. accumbens septi N. caudatus Tuberculum olfactorium Cortex cerebri Hippocampus dorsalis Hippocampus ventralis Gyrus dentatus Subiculum Cortex entorhinalis Cortex piriformis Cortex cingutaris Cortex frontalis
111 95 201 126
± ± ± ±
SHR (8)
8.3 7.1 16.3 9.3
65-+4.3 53 ± 3.6 55 ± 3.8 57 ± 3.9 52 ± 3.6 163 ± 11.5 751 - 62.3 769 ± 54.8 711 ± 47.6 701 -+ 46.5 77 ± 5.1 59 ± 2.8 110±7.5 64 ± 4.8 869 ± 63.2 159 ± 10.4 133 ± 9.4
71 69 80 71
± ± ± ±
5.2 4.4 6.2 5.3
Day 7
%change
WKY(8)
SHR(8)
± 7.4 ±7.6 ± 14.2 ± 10.7
114 105 230 168
± ± ±
7.0 7.6 16.4 10.3
115 96 217 157
_ 8.3 ± 8.1 __. 17.4 ± 11.3
61 ± 4 . 0 54 ± 4.2 49 ± 3.7 58 ± 4.1 54 ± 4.5 169 ± 10.2 763 ± 49.5 795 ± 61 3 745 ± 36.7 722 ± 57.3 75 ± 6.8 61 _ 5.3 115±8.1 61 ± 4.9 892 ± 66.5 153 ± 11.3 142 ± 10.8
78 58 64 70 58 190 811 860 811 824 81 64 112 74 921 190 151
+ ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.1 4.0 4.9 4.7 3.3 11.4 65.1 70.5 55.3 56.7 5.7 3.3 7.4 5.5 63.9 13.4 15.2
84 55 61 76 60 182 802 837 822 815 81 64 121 71 905 203 144
_ ± ± _ ± ± ± ± ± ± ± ± _ ± ± ±
5.7 3.7 4.8 4.9 4.6 11.5 58.2 58.1 48.5 57.6 5.5 4.4 8.3 5.9 60.8 15.6 10.3
85 74 95 81
± ± ± ±
5.1 4.8 4.9 3.7
86 75 91 84
± ± ±
5.2 4.1 6.0 5.8
115 91 190 139
69 68 85 71
± ± ± ±
4.8 5.2 6.8 5.2
* P < 0.01 compared with corresponding controls - ~ 20 pmol/g.
theter implanted chronically into the aorta near the orifice of the renal artery via the femoral artery and heart rate was triggered by blood pressure pulses36. Mean aortic blood pressures of female SHR and WKY before copulation were 178 _ 5 mm Hg (means _ S.E.M., n = 5)and 112 _ 4 mm Hg, respectively. For statistical analysis, Student's t-test was employed. RESULTS
Embryonic days 14 and 18 On embryonic days, localizations of cerebral ME and receptor binding were much more limited than those in the postnatal rat as shown below. At the embryonic developmental stage im-
munoreactive ME levels were found in 10 nuclei out of 83 cerebral nuclei and areas, particulary in the nucleus (n.) amygdaloideus centralis, n. periventricularis, n. supraopticus, n. interpeduncularis and n. suprachiasmaticus (Table I). Between embryonic days 14 and 18, ME levels increased by 20-40%. On embryonic day 14, [3H]ME receptor binding levels were found in the above former 4 nuclei and additional 5 nuclei on the embryonic day 18 (Table II). During the embryonic period there was no difference in these values between corresponding nuclei of SHR and WKY embryos (Tables I and II).
26 T A B L E IV
Methionine enkephalin receptor binding in cerebral nuclei of neonates of 14/KY and SHR in which [3H]ME receptor binding was not detected in the embryonic brains D a t a ( p m o l / g ) are m e a n s +_ S.E. for 5 animals.
Structure
Postpartum day 0
%change
day 7
% change
WKY(5)
SHR(5)
WKY(5)
SHR(5)
Rhombencephalon and mesencephalon N. intercalatus N. c o m m i s s u r a l i s Areapostrema N. tractus solitarii (caudal) N.originisn.hypoglossi N.dorsalisn.vagi N. olivaris inferior N. p a r a b r a c h i a l i s dorsalis N. parabrachialis ventralis Substantia nigra Substantia grisea centralis
65 306 129 542 210 225 748 726 386
± ± ± ± ± ±
± ± _ ± ± ±
81 351 146 628 262 281
84 374 138 592 248 293
± 33.7
71 322 116 567 215 237 792 708 398
Thalamus and epithalamus N. p a r a m e d i a n u s N.centromedianus N. d o r s o m e d i a l i s N. v e n t r o m e d i a l i s N. t r a c t u s s p i n a l i s n. trigemini T r a c t u s s p i n a l i s n . trigemini N. h a b e n u l a e medialis N. h a b e n u l a e lateralis
68 71 65 61 186 221 97 103
± ± ± ± ± ± ± ±
7.1 6.5 5.7 5.1 14.7 18.5 8.4 9.6
F o r m a t i o reticularis N. r a p h e o b s c u r u s N. r a p h e p a l l i d u s N.raphemagnus N. t e g m e n t i dorsalis p a i n N. t e g m e n t i ventralis N. l i n e a r i s m e s e n c e p h a l i N. reticularis centralis N. reticularis gigantocellularis N. reticularis paragigantocellularis N. r u b e r m e s e n c e p h a l i N. reticularis parvocellularis N. reticularis medialis N. reticularis dorsalis S u b s t a n t i a reticularis m e s e n c e p h a l i Z o n a incerta N. intralaminalis N. reticularis t h a l a m i
158 146 187 718 381 145 36 39 38 97 90 61 37 51 44 -
Hypothalamus N. posterior h y p o t h a l a m i N. corporis m a m m i l l a r i s medialis N. corporis m a m m i l a r i s lateralis N. v e n t r o m e d i a l i s N. d o r s o m e d i a l i s Arealateralishypothalami A r e a anterior h y p o t h a l a m i N. paraventricularis Median eminence lnfundibulum
5.8 25.9 11.3 48.6 20.6 19.4
5.2 21.8 10.5 53.1 18.3 21.7
_ 6.7 ___ 29.6 ± 12.5 ± 51.3 ± 25.2 ± 24.6
± ± ± ± ± ±
6.2 33.8 14.9 48.6 22.7 27.3
± 71.2 ± 60.5
853 --- 62.1 862 ± 73.0
829 ± 53.6 892 ± 65.9
± 31.3
462 ± 41.5
436 ± 38.2
60 63 72 64 132 240 91 111
± ± ± ± ± ± ± ±
6.4 5.7 5.4 6.2 10.7" 21.6 9.3 8.7
85 ± 8.4 89 ± 7.6 84±7.1 74 ± 8.3 226 ± 21.5 267 ± 25.8 105 ± 8.1 126 ± 10.2
88 ± 7.3 95 _ 8.9 85±6.4 78 ± 7.4 151 ± 24.3* 277 ± 21.8 111 ± 8.5 131 ± 9.7
± 14.5 ± 12.1 ± 15.4 ± 66.2 ± 24.3 ± 11.4 ± 4.2 ± 3.5 ± 3.0 ± 5.8 ± 7.5 __. 5.8 ± 2.4 ± 4.3 ± 4.2
167 149 176 707 352 130 30 33 35 90 81 66 31 58 47 -
± ± ± ± ± _ ± ± ± ± ± ± ± ±
13.2 13.6 16.9 68.4 31.7 12.7 2.1 2.8 3.3 8.7 8.4 6.3 3.5 5.6 3.9
189 173 211 816 452 186 51 48 42 113 104 80 48 72 53
± 16.7 _ 15.1 ___ 18.4 ± 65.0 ± 38.9 __. 18.2 ± 4.8 ± 3.2 ± 3.9 ± 10.5 ± 9.6 ± 6.5 ± 4.1 ± 6.9 ± 4.3
193 170 219 791 473 172 55 53 44 118 112 87 42 64 58
± ± ± ± ± ± ± ± ± ± ± ± ± ±
18.1 15.8 22.3 73.9 45.8 16.5 3.6 3.1 3.6 9.7 10.7 7.2 3.4 6.7 3.7
68 ± 5.4 45 ± 4.2 41 _ 4.3 65 ± 7.2 68 ± 5.4 136 ± 10.5 1 3 0 ± 11.5 403 __+ 38.7 41 ± 3.6
61 49 48 60 62 139 111 385 44
± ± _ ± ± ± ± ± ±
6.7 4.8 5.6 5.8 7.4 14.2 12.7 30.3 4.5
81 56 58 78 81 162 158 462 62
_ _ _ + + ± _
87 58 64 80 75 175 142 481 63
+ ± _ _ ± _ ± _ _
7.1 4.9 5.3 7.6 7.0 15.8 13.9 44.7 4.7
_ 65.3 ± 61.8
-29
7.5 4.1 5.2 8.0 6.4 13.3 11.8 41.3 5.2
-33
27 Table IV (Continued)
Structure
Postpartum
a~yo Fasciculus medialis telencephali (caudal) Fasciculus medialis telencephali (rostral) Fasciculus longitudinalis dorsalis Forel H Telencephalon N. amygdaloideus lateralis N. amygdaloideus intercalatus N. amygdaloideus basalis pars lateralis N. amygdaloideus basalis pars medialis N. amygdaloideus corticalis N. amygdaloidea anterior N. tractusolfactoriuslateralis N. proprius striae terminalis N. interstitialis striae terminalis dorsalis N. interstitalis striae terminalis centralis N. interstitialis striae terminalis medullaris N. dorsalis septi N. medialis septi N. lateralis septi N. intermedius septi N.accumbenssepti N. caudatus Tuberculum olfactorium Cortex cerebri Hippocampus dorsalis Hippocampus ventralis Gyrus dentatus Subiculum Cortex entorhinalis Cortex piriformis Cortex cingularis Cortex frontalis
%change
a~y7
%change
WK Y (5)
SHR (5)
WK Y (5)
SHR (5)
75 61 143 85
6.2 5.1 12.0 7.6
83 65 156 81
± ± ± ±
85 ± 71 ± 160 ± 91 ±
91 77 158 94
71 ± 7.5 48 _ 3.7 41 ± 3.6 40 ± 3.5 44 ± 3.2 38 __- 3.0 111 ± 10.4 516 ± 48.5 486 ~ 41.9 389 ± 42.1 368 ± 33.5 51 ± 3.8 42 ___3.4 85 ± 7.3 42 ± 3.3 586 ± 42.7 107 ± 6.5 86 ± 7.6
64 52 44 38 42 35 118 482 463 421 362 58 44 92 45 601 113 92
± 6.9 ± 4.5 ± 4.2 _.+ 3.1 ± 3.3 ± 4.2 ___ 10.9 ± 42.7 ± 37.5 ± 40.3 ± 34.8 ± 5.3 ± 3.9 ± 8.2 ± 4.1 ± 59.6 ± 8.5 ± 6.7
-
45 51 63 55
± ± ± ±
~ 4.1 -_- 4.5 ± 4.8 ± 4.1
5.8 4.1 11.3 6.9
7.1 7.0 11.5 7.2
84 ± 8.0 62±4.1 49 ± 4.3 51 ± 4.3 48 ± 5.0 45 ± 4.2 141 ± 12.5 623 ± 51.3 548 ± 49.2 485 - 42.3 40~ ± 41.7 74 ± 6.2 52±4.1 113___11.5 52 ± 2.6 715 ± 50.2 132 ± 8.7 103 ± 8.1
-
-
___3.8 ± 4.2 ± 5.2 ± 3.7
6.4 6.2 13.2 7.5
91 ± 7.7 66±4.9 55 ± 4.7 47 ± 3.8 55 ± 5.2 47 ± 3.6 132 - 10.6 605 ± 55.7 581 ± 47.0 472 ± 40.5 422 ± 38.8 70 ± 7.3 55±5.2 107±11.1 52 ± 4.8 741 ± 52.8 122 _ 10.9 115 ± 8.5
m
_
41 55 65 59
± ± ± ±
i
60 63 71 65
_ 3.4 ± 4.9 ± 5.5 ±4.8
57 ± 65 ± 77 ± 67±
4.1 5.2 5.8 5.1
* P ~ 0.01 compared with corresponding controls - < 10 pmol/g.
Postnatal days 0 and 7 At the day o f birth, i m m u n o r e a c t i v e M E levels a n d M E r e c e p t o r b i n d i n g in 10 a n d 9 n u c l e i respectively increased rapidly w h e r e a s h a r p 2 - 3 fold i n c r e a s e w a s n o t i c e d . Both l e v e l s a b r u p t l y a p p e a r e d in 65 o u t o f 73 n u c l e i in w h i c h b o t h v alues w e r e n o t d e t e c t a b l e d u r i n g t h e p r e n a t a l p e r i o d ( T a b l e s III a n d IV). B o t h l e v e l s i n d i c a t e d the a d u l t p a t t e r n a n d w e r e p a r t i c u l a r l y h i g h in the n. t r a c t u s s o l it a r i i ( c a u d a l part), n. p a r a b r a chialis, n. a c c u m b e n s s e p t i a n d g r o u p o f t h e stria t e r m i n a l n u c l e i . B e t w e e n p o s t n a t a l d a y s 0 a n d 7,
most nuclei on the postnatal day 7 were higher than those at 4 and 20 weeks of age 32. During the neonatal period, SHR had lower ME levels in the tractus spinalis nervi trigemini (Vts) and ME receptor binding in the n. tractus spinalis nervi trigemini (Vnts) than those of WKY (Fig. 1). The difference persisted in young SHR at the age of 4 weeks and disappeared in adult SHR at age of 20 weeks32. A ntinociceptive threshold T a b l e V s h o w s t h e d i f f e r e n c e in a n t i n o c i c e p -
both M E levels and r e c e p t o r b i n d i n g increased
tive s e n s i t i v i t y o f n e o n a t a l , y o u n g a n d
by 5-20% a n d 2 0 - 4 0 % , r e s p e c t i v e l y . T h e a b s o lute v a l u e s o f M E l e v e l s a n d r e c e p t o r b i n d i n g in
SHR a n d W K Y . S l i g h t b u t s i g n i f i c a n t d i f f e r -
adult
e n c e s c o u l d b e d e m o n s t r a t e d in all a n i m a l s . In
28 Tractus s P l n o l l s nervl t r l o e m l n l
400
I
blrtn
~~o t ~ ~ ~ ~EL 0 WKY • SHR
~
tained in y o u n g S H R (4 weeks old) by the hotplate method. However, adult S H R (20 weeks old) showed a relative antinociceptive effect when c o m p a r e d with normotensive W K Y o f the same age in both tests (Table V).
MERB
200 "
DISCUSSION
P~O,05
100
100 '
a
001"4
D-~
LI
I
l
4
20
blrth~
|1
-I
Nucleus troctus sotnolls
0
i
I
1
•
x.
20
nervl trlg~lnl
400
~E IL I
Evidence presented in this p a p e r indicates m a r k e d heterogeneity o f silver grain distribution in various brain areas and sharp boundaries o f grain densities in certain areas, which indicate that observed localizations o f both M E levels and M E receptor binding are not d e t e r m i n e d by diffusions but represent the distribution o f both levels. F u r t h e r m o r e , the possibility that [3H]decomposed product may bind to non-selective sites can be excluded, since regional selectively o f grain and extent o f grain densities are in accordance with those o f M E levels. Non-specific binding a m o u n t s to a b o u t 3% o f the total binding in most nuclei. Our results indicated that the regional distribution o f i m m u n o r e a c t i v e M E and its receptor binding could be detected respectively in 10 and 4 o f 83 cerebral nuclei on the e m b r y o n i c day 14, and also 10 and 9 nuclei on the e m b r y o n i c day 18, 3 days before birth. This indicates that the prenatal d e v e l o p m e n t o f cerebral M E neurons is limited to some nuclei and that M E receptor systems in the prenatal period develop later than ME i m m u n o r e a c t i v e cells. During the prenatal period of d e v e l o p m e n t in these limited nuclei, the levels o f M E and receptor binding increased at a higher rate than those in the early postnatal period. T h e finding is consistent with the pre-
MERB birth
g ~.200 100
0 01--~ LI
i
-
0 i
M,
1
q
20
Time In
-I
weeksafterDtrtn
0
I
tl
20
Fig. 1. Development of methionine-enkephalin-like immunoreactivity (MELI) and [3H]methionine-enkephalin receptor binding (MERB) in the Vts and Vnts of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Values are expressed as means --. S.E.M. for 8 animals per group. Data for both animals of 4 and 20 weeks after birth are those as described previously32.
both animal groups o f 1 and 4 weeks o f age, antinociceptive thresholds o f S H R were slightly but significantly (P < 0.05) lower in the tail-flick test than those o f W K Y . Similar results were obTABLE V
A ntinociceptive thresholds in male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats ( W K Y)
The data represent means ± S.E.M. for the number of animals shown in parenthesis. A ge weeks
n
1 4 20
9 15 16
* P < 0.05.
** P < 0.01.
Tailflick (latency, s)
Hot-plate (latency, s)
WK Y
SHR
% difference
WK Y
SHR
% difference
1.48 -4-0.054 3.02 ± 0.205 3.03 ± 0.219
1.33 ± 0.0372* 2.53 ± 0.114" 5.29 ± 0.728**
- 11 -16 +75
n.d. 11.8 ± 0.972 9.84 ± 0.974
n.d. 9.04 ± 0.755* 17.3 +__1.93"*
23 +76
29 vious reportss,9 that there is an initial appearance of [3H]opiate antagonist binding from embryonic day 15 through birth followed by a slower increase to adult levels. The localization of both levels in most nuclei which abruptly appeared on the day of birth indicated the adult pattern. There was no rank order correlation between both levels presented in the prenatal and postnatal periods. This process of the delayed detection of immunoreactive ME is quite in parallel to that of immunoreactive LE in rat embryonic brain regions in which endorphin maturation is much faster2. The abrupt appearance of ME and receptor binding of neonatal rats coincides closely with an abrupt increase in the pain thresholds in dams 1-2 days before parturition. This antinociceptive action is present only during pregnancy and is abolished by naltrexone administration ~4. The full-term human placenta contains immunoreactive ME and fl-endorphin of which the local release may regulate sensory transmission from the uterus and vaginal tract during childbirth41. During the embryonic and neonatal periods of 4 developmental time-points we studied, ME receptor binding in various nuclei was the highest on the postnatal day 7. The results are consistent with the previous findings of a rapid increase from birth to weaning of [3H]ME receptor39,50,57 and [3H]opiate agonist and antagonist receptors8.9,38,57. Such early postnatal development of ME neurons may be be responsible for neonatal withdrawal symptoms in infants of addicted mothers5°. During the period of the rat neonatal days 0 and 7, ME levels in the Vts and ME receptor binding in the Vnts in the medulla oblongata were markedly lower in SHR than in WKY. The difference persisted in young SHR at age of 4 weeks but not in adult SHR at age of 20 weeks32. The Vnts caudal and rostral 5 known to receive afferent terminals from the facial and oral areas, to send both ascending fibers to the ventral thalamus 13.44 and descending fibers to the spinal cord 7,42,49, thus modulating sensory impulses in the spinal dorsal horn. Particularly in the rodent, the Vnts receives afferent inputs from the mystacial vibrissae ~L'2, which are the important sniff-
ing-linked tactile sensory organs in the rat 52. Since nociceptive thresholds of neonatal SHR determined presently by two methods were lower than those of neonatal WKY, noxious sensory perception of SHR neonates might be greater than that of WKY neonates. Although decreased reactivity to noxious stimuli has been reported in SHR 53 and in rats with raised blood pressure J°,55.56, these animals are adult or young but not neonate. More reckless and hyper-reactive behavior of neonatal SHR apparently heightened pain sensitivity in the present study. However, adult SHR exhibited an antinociceptire effect when compared with normotensive adult SHR, in agreement with rats with raised blood pressure 1°.55.56. Thus, the decreased ME and receptor binding in the Vts and Vnts of SHR neonates probably disinhibit afferent noxious as well as non-noxious sensory information. Such a genetic deficiency might then produce effects on the proper development of the CNS system, similar to the deleterious effects of sensory deprivation on the ontogenetic development of the visual system. In young SHR, prolonged deprivation of such environmental stimuli as social contacts and confrontations 15 and light28 attenuates and delays the rise of the mean blood pressure as paralleled by less pronounced cardiovascular structural changes. Furthermore, electrical stimulation of the Vts and Vnts area at the level of the obex is known to produce hypotension and bradycardia in decerebrate or anesthetized rabbits, which is termed the trigeminal depressor response (TDR) 26. Its hemodynamic pattern is similar to aortic depressor reflex25. In view of rich presence of ME immunoreactivity and receptor in the area, the participation of ME neuronal activity with the TDR is highly probable. In SHR neonates, the average arterial blood pressure is very small but already significantly higher than that in WKY 6,17. In SHR, the neonatal deficiency of ME neurons in the Vts and Vnts was also confirmed to precede with the following expansive abnormality in 4 weeks of age: the decreased ME levels and receptor binding in the n.reticularis lateralis, n. arcuatus and n. corporis mammillaris, and the increased ME levels and receptor binding in
30 the n. dorsalis nervi vagi, n. amygdaloideus medialis and centralis, and group of the stria terminal nuclei 32'33. The ME neuronal activation in these latter nuclei is consonant with the previous finding of the enhanced glucose utilization rate in the same nuclei ~7. In young SHR, there is preganglionic-dependent activation of sympathetic nervous system 35,36, and central hyperreactivity accompanied by a greater sympathetic activation 16,36.These results suggest that there is an ontogenic correlation between enhanced sensory perception and sympathetic activation. In conclusion, at the day of birth ME neurons
abruptly appeared in most cerebral nuclei except for several nuclei already present in the prenatal period. During the perinatal period of SHR, ME levels in the Vts and ME receptor binding in the Vnts were markedly lower in SHR than WKY neonates. The neonatal ME neuronal deficiency in these areas leading to further ME abnormality in the hypothalamus, strio-amygdaloid complex and n. dorsalis nervi vagi 32 may be prerequisite for the development of sympathetic activation and hyper-reactivity in young SHR.
REFERENCES
12 Erzurumlu, R. S. and Killackey, H. R., Order in the developing rat trigeminal nerve, Develop. Brain Res., 3 (1982) 30:%310. 13 Fukushima, T. and Kerr, F. W. L., Organization of trigemino-thalamic tracts and other thalamic afferent systems of the brainstem in the rat: presence of gelatinosa neurons with thalamic connections, J. comp. Neurol., 183 (1979) 169- 184. 14 Gintzler, A. R., Endorphin-mediated increases in pain threshold during pregnancy, Science, 210 (1980) 193 195. 15 Hallb~ick, M., Consequence of social isolation on blood pressure, cardiovascular reactivity and design in spontaneously hypertensive rats, A cta physiol, stand., 93 (1975) 455-465. 16 Hallb/ick, M. and Folkow, B., Cardiovascular responses to acute mental 'stress' in spontaneously hypertensive rats, A ctaphvsiol, scand., 90 ( t 974) 684-698. 17 Hayashi, T. and Nakamura, K.. Activation of cerebral neuronal activity in spontaneously hypertensive rats as devaonstrated by the 14C-deoxyglucose method, NaunvnSchmiedeberg's Arch. Pharmacol., 316 ( 1981 ) 331- 339. 18 Heybach, J. P. and Danellis, J. V., The effect of pituitaryadrenal function in the modulation of pain sensitivity in the rat, J. Physiol. (Lond.), 283 (1978) 331- 340. 19 H6kfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in the central neurons system, Neurosci. Lett., 5 (1977) 25-31. 20 H6kfelt, T., Terenius, L., Kuypers, H. G. J. M. and Dann, O., Evidence for enkephalin immunoreactive neurons in the medulla oblongata projecting to the spinal cord, Neurosci. Lett., 14 (1979) 55- 60. 21 Hong, J. S., Yang, H.-Y. T., Eratta, W. and Costa, E., Determination ofmethionine enkephalin in discrete regions of rat brain, Brain Research, 134 (1977) 383- 386. 22 Hughes, J., Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine, Brain Research, 88 (1975) 295- 308. 23 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 (1976) 577~ 579.
1 Bardo, M. T., Bhatnagar, R. K. and Gebhart, G. F.. Opiate receptor ontogeny and morphine-induced effects: influence of chronic footshock stress in preweanling rats, Develop. Brain Res., 1 ( 1981 ) 487-495. 2 Bayon, A., Shoemaker, W., Bloom, F. E., Mauss, A. and Guillemin, R., Perinatal development of the endorphinand leucine enkephalin-containing systems in the rat brain, Brain Research, 179 (1979) 93-101. 3 Bellet, M., Elghozi, J. L., Meyer, P., Pernolett, M. G. and Schmitt, H., Central cardiovascular effects of narcotic analgesics and enkephalins in rats, Brit. J. Pharmacol., 71 (1980) 365-369. 4 Bolme, P., Fuxe, K., Agnati, L. F., Bradley, R. and Smythies, J., Cardiovascular effects of morphine and opioid peptides following intracisternal administration in chloralose-anesthetized rats, Europ. J. Pharmacol., 48 (1978) 319--324. 5 Broton, J. G. and Rosenfeld, J. P., Rostral trigminal projections signal perioral facial pain, Brain Research, 243 (1982) 395-400. 6 Bruno, L., Azar, S. and Weller, D., Absence ofa pre-hypertensive stage in post-natal Kyoto hypertensive rats, Jap. HeartJ., 20 Suppl. I (1979) 90-92. 7 Burton, H., Craig, A. D., Poulos, D. A. and Molt, J. T., Efferent projections from temperature sensitive recording loci within the marginal zone of the nucleus candalis of the spinal trigeminal complex in the cat, J. comp. Neurol., 183 (1979) 753-778. 8 Clendeninn, N. J., Petraitis, M. and Simon, E. J., Ontological development of opiate receptors in rodent brain, Brain Research, 118 (1976) 157-160. 9 Coyle, J. T. and Pert, C. B., Ontogenetic development of [3H]naloxone binding in rat brain, Neuropharmacol., 15 (1976) 555- 560. l0 Dworkin, B. R., Filewich, R. J., Miller, N. E., Craigmyle, N. and Pickering, T. G., Baroreceptor activation reduces reactivity to noxious stimulation: implications for hypertension, Science, 205 (1979) 1299-1301. 11 Erzurumlu, R. S., Bates, C. A. and Killackey, H. P., Differential organization of the thalamic projection cells in the brain stem trigeminal complex of the rat, Brain Research, 198 (1980) 427-433.
31 24 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, R. E. Krieger, New York, 1970. 25 Kumada, M., Dampney, R. A. L., Whitnall, M. H. and Reis, D. J., Hemodynamic similarities between the trigeminal and aortic vasodepressor responses, Amer. J. Physiol., 234 (1978) H6%H73. 26 Kumada, M., Dampney, R. A. L. and Reis, D. J., The trigeminal depressor response: a novel vasodepressor response originating from the trigeminal system, Brain Research, 119 (1977) 305-326. 27 Kuraishi, Y., Sugimoto, M., Hirota, N. and Takagi, H., Lack of effect of morphine and noxious stimuli on the Met-enkephalin content in the spinal dorsal horn and nucleus reticularis gigantocellularis of the rat, Life Sci., 30 (1981) 2071-2077. 28 Lais, L. T., Bhatnagar, R. A. and Brody, M. J., Inhibition by dark adaptation of the progress of hypertension in spontaneously hypertensive rat (SHR), Circulat. Res., 34 and 35, Suppl. I (1974) 155-160. 29 Laubie, M. and Schmitt, H., Indication for central vagal endorphinergic control of heart rate in dogs, Europ. J. Pharmacol., 71 (1981)401-409. 30 Laubie, M., Schmitt, H., Vincent, M. and Resmond, G., Central cardiovascular effects of morphinomimetic peptides in dogs, Europ. J. Pharmacol., 46 (1977) 67-71. 31 Lord, J. A. H., Waterfield, A. A., Hughes, J. and Kosterlitz, H. W., Endogenous opioid peptide: multiple agonists and receptors, Nature (Lond.), 267 (1977) 495-499. 32 Nakamura, K. and Hayashi, T., Methionine enkephalinergic neuronal activity in cerebral nuclei of spontaneously hypertensive rats, Hypertension, 4 (1982) 662669. 33 Nakamura, K. and Hayashi, T., Altered methionine enkephalin immunoreactivity in cerebral nuclei of spontaneously hypertensive rats, Clin. exp. Hyp., A4 (1982) 297-301. 34 Nakamura, K., Kuntzman, R., Maggio, A. C., Augulis, V. and Conney, A. H., Influence of 6-hydroxydopamine on the effect of morphine on the tail-flick latency, Psychopharmacol., 31 (1973) 177-189. 35 Nakamura, K. and Nakamura, K., Selective activation of sympathetic ganglia in young, spontaneously hypertensive rats, Nature (Lond.), 266 (1977) 265-266. 36 Nakamura, K. and Nakamura, K., Role of brainstem and spinal noradrenergic and adrenergic neurons in the development and maintenance of hypertension in spontaneously hypertensive rats, Naunyn-Schmiedeberg's Arch. Pharmacol., 305 (1978) 127-133. 37 Palkovits, M. and Jacobowitz, D. M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain - II.Hindbrain (mesencephalon, rhombencephalon), J. comp. Neurol., 157 (1974) 2% 42. 38 Pasternak, G. W., Zhang, A. and Tecott, L., Developmental differences between high and low affinity opiate binding sites: their relationship to analgesia and respiratory depression, Life Sci., 27 (1980) 1185-1190. 39 Patey, G., Baume, S., Gros, C. and Schwartz, J., Ontogenesis of enkephalinergic systems in rat brain: postnatal changes in enkephalin levels, receptors and degrading enzyme activities, Life Sci., 27 (1980) 245 252. 40 Petty, M. A. and Reid, J. L., Opiate analogs, substance P and baroreceptor reflexes in the rabbit, Hypertension, 3 Suppl. I (1981) 142- 147. 41 Rama Sastry, B. V., Barnwell, S. L., Tayeb, O. S., Janson,
V. E. and Owens, L. K., Occurrence of methionine enkephalin in human placental villus, Biochem. Pharmacol., 29 (1980) 475-478. 42 Ruggiero, D. A., Ross, C. A. and Reis, D. J., Projections from the spinal trigemlnal nucleus to the entire length of the spinal cord in the rat, Brain Research, 225 (1981) 225233. 43 Schaz, K., Stock, G., Simon, W., Schl6r, K.-H., Unger, T., Rockhold, R. and Ganten, D., Enkephalin effects on blood pressure, heart rate, and baroreceptor reflex, Hypertension, 2 (1980) 395- 407. 44 Shigenaga, Y., Takabatake, M., Sugimoto, T. and Sakai, A., Neurons in marginal layer of trigeminal nucleus caudalis projecting ventrobasal complex (VB) and posterior nuclear groups (PO) demonstrated by retrograde labeling with horseradish peroxidase, Brain Research, 166 (1979)391 396. 45 Simantov, R., Kuhar, M. J., Uhl, G. R. and Snyder, S. H., Opioid peptide enkephalin: immunohistochemical mapping in rat central neurons system, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 2167-2171. 46 Simantov, R. and Snyder, S. H., Morphine-like factors in human brain: structure elucidation and interactions with the opiate receptor, Proc. nat. Acad Sci. U.S.A., 73 (1976) 2515-2519. 47 Smith, T. W., Hughes, J., Kosterlitz, H. W. and Sosa, R. P., Enkephalins: isolation, distribution and function. In H. W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides, North-Holland, Amsterdam, 1977, pp. 5~62. 48 Stengaard-Petersen, K. and Larsson, L.-I., Comparative immunocytochemical localization of putative opioid ligands in the central neurons system, Histochemistry, 73 (1981)8%114. 49 Tohyama, M., Sakai, K., Salvert, D., Touret, M. and Jouvet, M., Spinal projections from the lower brain stem in the cat as demonstrated by the horseradish peroxidase technique. I. Origins of the reticulospinal tracts and their funicular trajectories, Brain Research, 173 (1979) 383403. 50 Tsang, D. and Ng, S.C., Effect of antenatal exposure to opiates on the development of opiate receptors in rat brain, Brain Research, 188 (1980) 199-206. 51 Uhl, G. R., Goodman, R. R., Kuhar, M. J., Childers, S. R. and Snyder, S. H., Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Research, 166 (1979) 75-94. 52 Welker, W. I., Aalysis of sniffing of the albino rat, Behaviour, 22 (1964) 223-244. 53 Wendel, O. T. and Bennett, B., The occurrence of analgesia in an animal model of hypertension, Life Sci., 29 (1981) 51~521. 54 Yang, H.-Y., Hong, J. S. and Costa, E., Regional distribution of Leu- and Met-enkephalin in rat brain, Neuropharmacol., 16 (1977) 303-307. 55 Zamir, N. and Segal, M., Hypertension-induced analgesia: changes in pain sensitivity in experimental hypertensive rats, Brain Research, 160 (1979) 170- 173. 56 Zamir, N. R., Simantov, R. and Segal, M., Pain sensitivity and opioid activity in genetically and experimentally hypertensive rats, Brain Research, 184 (1980) 299- 310. 57 Zhang, A.-Z. and Pasternak, G. W., Ontogeny of opioid pharmacology and receptors: high and low affinity site differences, Europ. J. PharmacoL, 73 (1981) 29- 40.