Development of opioid systems: peptides, receptors and pharmacology

Brain Research Reviews, 12 (1987) 397-421

397

Elsevier BRR 90070

Development of opioid systems: peptides, receptors and pharmacology Julia McDowell and Ian Kitchen Department of Biochemistry, Division of Pharmacology and Toxicology, University of Surrey, Guildford, Surrey (ll. K.)

(Accepted 7 April 1987) Key words: Opioid; Ontogeny; Opioid peptide; Opioid receptor; Opioid pharmacology; Antinociception;

Perinatal

CONTENTS 1. Introduction

.............................................................................................................................................

398

2. Opioid peptide ontogeny ............................................................................................................................. 2.1. Introduction ....................................................................................................................................... 2.2. Proopiomelanocortin products ............................................................................................................... 2.3. Proenkephalin products ........................................................................................................................ 2.4. Prodynorphin products .........................................................................................................................

398 398 399 402 403

3. Receptor ontogeny ..................................................................................................................................... 3.1. Introduction ....................................................................................................................................... 3.2. Receptor ontogeny in the rat: non-selective ligands ...................................................................................... 3.3. Receptor ontogeny in the rat: selective ligands ............................................................................................ 3.4. Receptor ontogeny in other species .......................................................................................................... 3.5. Peripheral receptor ontogeny .................................................................................................................

403 403 406 406 407 408

4. Pharmacology of the opioid system during ontogeny ........................................................................................... 4.1. Introduction ....................................................................................................................................... 4.2. Antinociception .................................................................................................................................. 4.3. Locomotor activity .............................................................................................................................. ...................................................................................................................... 4.4. Opioid pharmacokinetics 4.5. Toxicity ............................................................................................................................................ 4.6. Respiration and cardiovascular system ...................................................................................................... 4.7. Isolated tissues .................................................................................................................................... 4.8. Genotype variations ............................................................................................................................. 4.9. Miscellaneous .....................................................................................................................................

408 408 408 408 409 409 409 411 411 412

5. Modulation of the ontogeny of the opioid system ................................................................................................ 5.1. Introduction ....................................................................................................................................... 5.2. Opioid administration .......................................................................................................................... 5.3. Stress ............................................................................................................................................... 5.4. Non-opioid drugs ................................................................................................................................ 5.5. Toxic chemicals ...................................................................................................................................

412 412 412 415 416 416

.................................................................................................................................

6. Conclusions/summary Acknowledgements References

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. ,

Correspondence: I. Kitchen, Department ford, Surrey GU2 5XH, U.K.

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416

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417

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417

of Biochemistry, Division of Pharmacology and Toxicology, University of Surrey, Guild-

398 2. OPIOID

1. INTRODUCTION

As with other neurochemical standing of endogenous opioid

systems the underontogeny has been

preceded by a good deal of experimental study in the adult. Application of methodology established for the adult is often difficult in the neonate and in this respect endogenous opioids are no exception. Nevertheless, the enormous scientific effort that was made in this area in the late 1970’s resulted tion of ontogenetic

in the publica-

studies from several laboratories

by 1980, only 5 years after the initial isolation of a morphine like substance from pig brain56. This review covers the literature on ontogeny of the opioid system since 1976 and in addition includes earlier whole animal studies on the ontogeny of morphine antinociception and toxicity. The review covers the ontogeny of opioid peptides, receptors and pharmacology. Studies of a histological nature and ontogeny of opioids in tissue culture are beyond the scope of the review but it should be recognised that there is also extensive literature in these areas. Ontogenetic studies have yielded information on interrelationships between opioids and various physiological functions such as peripheral and central pain perception, behavioural control by the limbic system and neuroendocrinological control. Much experimental work has been directed at determination of levels of endogenous opioids and the distribution and kinetic parameters of opioid receptor binding. In addition the link between opioid ontogeny and behavioural and physiological development in the whole animal has been studied using various pharmacological tests. It should be noted that there are several methodological constraints associated with developmental studies. Limitations imposed by the size of foetal and embryonic brains are particularly a problem in small animal species. In addition short gestation periods and rapid postnatal development in small animals necessitates accurate determination of the time of conception and birth. It is also worthy to note the differences in relative maturity between animal species during the perinatal period. For example the guinea pig at birth has its eyes open, exhibits many adult responses and in brain tissue cell proliferation is complete. In contrast the newborn rat is immature, eye opening occurring only after two weeks and cell proliferation

in the brain continuing

beyond this time.

PEPTIDE

ONTOGENY

2.1. Introduction As would be expected

levels of opioid peptides

in

the developing neonate are considerably less than in the adult and this has necessitated sensitive assay techniques. Some radioimmunoassays

of the early studies employed with sufficient sensitivity but rel-

atively poor selectivity. Thus results were expressed as enkephalin or endorphin equivalents. More recently ~p~c6,2"."O,lO".l27 or gel filtration chromatographys5.‘0”,‘2s combined with radioimmunoassay or bioassay 6~127have allowed

measurement

of specific

opioid peptides. This is extremely important since there may be up to 20 opioid peptides derived from the 3 opioid peptide precursor molecules serving distinct physiological functions (for review see ref. 73). In brief, there are 3 opioid precursors; proopiomelanocortin which produces P-endorphin; proenkephalin which is processed to yield 4 major enkephalin peptides, [Met]-enkephalin, [Leu]-enkephalin, [Met]enkephalyl-Arg6-Phe7 and [Met]-enkephalyl-Arg6Gly’-Leu*; and prodynorphin which is processed to various dynorphin peptides and a- and /3-neoendorphin all of which are extended sequences of [Leu]-enkephalin. It should also be noted that the sensitivity of the assay determines the limit for earliest detection since levels in foetal animals are extremely low. Expression of results is also important in the comparison of studies. Levels may be expressed per brain region, per gram wet weight of tissue or per milligram protein. Levels per brain region invariably increase with age until adulthood whereas concentrations per wet weight tissue or per milligram protein will vary and are dependent on the relative increases of both the opioid and the protein content of the brain region. This has the most marked effect on peak levels; if results are expressed per brain region then the peak is usually seen at the oldest age studied or in adult animals, whereas if results are expressed as a concentration the peak may occur at different times during ontogenesis. The majority of ontogenetic studies have been carried out in the rat central nervous system (CNS) (Table I). Foetal studies have confirmed that all opioids are present in the CNS well before birth. With the exception of the cerebellum, the peak level of en-

dogenous opioid peptides appears to occur during the

in the forebrain

third postnatal week, but as discussed previously is dependent on whether results are expressed

days and at 3 days in the cerebellum followed by decreases in content to adult values117*11g.In addition /?endorphin in the pituitary remained constant until the second postnatal week before increasing rapidly

this per

brain area or as a concentration. This progression of development is mirrored by the ontogeny of opioid receptors (see Section 3.). The early work of Garcin and Coyle38,3g showed the ontogeny of an endogenous morphine-like in the rat CNS which was capable of inhibiting

factor nalox-

one binding. At embryonic day 17 levels of this factor were 20% of that found in the adult increasing to 46% at birth. During the first postnatal week levels remained constant before increasing to adult values and showing

some parallel

to the receptor

devel-

opment. In the following years more selective assays have been employed to investigate the ontogeny of the various opioid peptides and this is described in the following sections. 2.2. Proopiomelanocortin products P-Endorphin can be detected as early as embryonic day 13 in rat whole braingl. Bayon et al.13 and Bloom et al.” measuring /3-endorphin in separate brain regions at embryonic day 16 found the distribution to be similar to the adult pattern although levels were only <2% of the amounts found in 25day-old animals. Histological studies have also demonstrated the presence of /?-endorphin immunoreactivity at embryonic day 14 in the spinal cord5’ though this immunofluorescence disappears abruptly by postnatal day 28, and is absent in the adult. Ng et al. ‘i demonstrated that /l-endorphin content of several brain areas increases with age, but absolute concentrations in the forebrain and hindbrain increased only minimally after embryonic day 18 which reflected large increases in the protein content of these areas. A similar observation has been made by other workers13,15; within the telencephalon, hippocampus and striatum levels of /3-endorphin increase less rapidly than protein concentrations between embryonic day 16 and postnatal day 6 and accordingly /3-endorphin levels are seen to fall. However, true decreases in /?-endorphin content have been shown in the mediobasal hypothalamus during the second postnatal week 53. Subsequent to these early postnatal fluctuations most workers have found that levels increase to adult values, with the exception of Tsang et al.“‘,“’ who reported peak levels of /3-endorphin

at 15 days, in the brainstem

to adult levels at day 20 (refs. 117, 119). Most recent studies have shown increases dorphin

immunoreactivity

in

the

at 20

in p-en-

hypothalamus,

amygdala, pons mid-brain, cortex and pituitary tween 10 and 80 days65. Only the hippocampal gions showed a decrease There are also indications

bere-

between 29 and 80 days. of a differential activity of

processing enzymes for proopiomelanocortin are both age and brain region dependent65.

which

Alessi et al.’ have demonstrated a differential development of /3-endorphin in the anterior and intermediate lobe of the pituitary. In contrast with the work of Tsang et al. ll’,*lg levels of /I-endorphin increased in both areas from birth to adulthood. HOWever, the rate of increase in the two areas varied after postnatal day 7, increasing more rapidly in the intermediate lobe than in the anterior lobe where levels did not rise until puberty (days 35-42). In addition N-acetylation of p-endorphin occurs in both lobes of the pituitary at birth’, but N-acetyl P-endorphin constitutes a much higher proportion of total endorphin in the intermediate lobe (70%) than in the anterior lobe (25%). The proportion in the anterior lobe decreases to adult levels of ~5% by day 42 whereas in the intermediate lobe N-acetyl /?-endorphin increases to 90% of total endorphin. Again, there is clearly a differential metabolic activation which is age dependent. As can be seen from the preceding discussion there are marked regional differences in the postnatal development of /3-endorphin and some disagreement between studies. The question of whether /?-endorphin develops in parallel with its putative receptor (the p-receptor) must remain open since binding studies with selective ligands show rapid postnatal development of this receptor to day 21 and on this point there is good agreement between laboratories (Section 3.3.). However, in the only study where /?-endorphin levels and receptor binding were monitored in parallel llg there was some accord in the developmental profile. Mechanisms for the stimulated release of B-endorphin are immature at birth. Spontaneous or potassi-

400 TABLE

I

Ontogenesis of opioid peptides in the rat CNS Opioid peptide

Age

Proenkephalinproducts Enkephalina

-6 to 25 days

1 to 30 days

10,21,30,40,50 and 100 days

[Met]-enkephalin

[Leu]-enkephalin

0, 1,2,3,4,5,6 and 7 weeks 0 to 28 days lo,21 and 30 days -2 and 1 day -6,-l, 14andadult -6, -2,6 and 25 days

0,4,7, 14,21,28 and adult lo,21 and 30 days 0,3,7,14,21 and 30 days, adult

[MetJ-enkephalylArg6-Phe’ [Metl-enkephalylArg6-Gly’-Leus Prodynorphin products Dynorphin Dynorphin,_,

Dynorphin,_,,

Dynorphin-B

a-Neoendorphin

Medulla and midbrain Diencephalon Telencephalon Striatum Ventral hippocampus Cortex and amygdala Forebrain Brainstem Cerebellum Striatum

25 days 25 days 25 days 25 days 25 days 25 days 10 days 7 days 7 days 21 days

Bloom et al.”

Tsang et al.‘”

Winder

3 weeks

Whole brain

et al.“’

Weissman

et al.“’

Striatum Striatum Hippocampus Whole brain minus cerebellum Medulla and midbrain

14 days 25 days

Diencephalon Midline telencephalon Striatum Ventral hippocampus Cortex and amygdala Striatum

25 25 25 25 25 28

Cortex Striatum Neurointermediate

21 days 21 days 21 days/adult

Bailey and Kitchen’ Seizinger et al. lo5

Dahl et al.‘” Bailey and Kitchenh

pituitary

21 to 28 days 21 days as adult

days days days days days days

Patey et al.yh Bailey and Kitchen6 Zamir et al.‘s’ Dahl et al.?? Bayon et al.”

Patey et al.4h

-6, - 1, 14 and adult lo,21 and 30 days

Anterior pituitary Whole brain minus cerebellum Striatum

30 days 14 days 21 days

lo,21 and 30 days -2, 1,7.14 days

Striatum Hippocampus

10 and 21 days 14 days

Bailey and Kitchen6 Zamir et al. “s

1,4,8,14,21 and 30 days 0,3,7,14,21 and 30 days, adult -2, 1,7,14days 0,3,7,14,21 and 30 days, adult

Neurointermediate

pituitary

30 days

Seizinger

et al.“‘6

Neurointermediate

pituitary

21 days

Seizinger

et al.

Hippocampus Neurointermediate

pituitary

14 days 30 days

Zamir et al.“i Seizinger et al. “I5

pituitary

2 1 to 30 days 14 days 21 days

Zamir et al.“’ Seizinger et al.“‘i

Anterior pituitary Hippocampus Neurointermediate

pituitary

21 days 14 days 30 days

Zamir et a1.13’ Seizinger et al. I”’

Neurointermediate

pituitary

30 days

Seizinger

30 days 14 days

Zamir

-2,1,7,14 days 0,3,7,14,21 and 30 days, adult -2,1,7,14 days 1.4,8,14,21 and 30 days 0,3,7,14,21 and 30 days, adult -2, 1,7,14days

Anterior pituitary Hippocampus Neurointermediate

Anterior pituitary Hippocampus

as adult

“15

et al.“”

et < nl.13’ (conrinued)

401 TABLE I (continued) Opioid peptide

/I-Neoendorphin

Proopiomelanocortin

Age

Brain region

0,3,7,14,21 and 30 days, adult -2,1,7,14 days

Neurointermediate

Peak levelsb

Reference

21 days

Seizinger et al. lo5

Hippocampus

14 days

Zamir et a1.i3’

Medulla and midbrain

25 days

Bayon et aLi

Diencephalon Midline telencephalon Striatum Ventral hippocampus Cortex and amygdala Medulla and midbrain Diencephalon Midline telencephalon Striatum Forebrain Brainstem Cerebellum Forebrain Brainstem Cerebellum Mediobasal hypothalamus Forebrain

25 days -6 and 25 days -6 days -6 days 25 days 25 days 25 days 25 days -6 days 15 days 21 days 2 to 3 days 15 days 21 days 2 to 3 days 28 days 3 months

Midbrain Hindbrain Hypothalamus Pituitary Anterior pituitary

3 months 3 months 3 months 3 months adult

Intermediate pituitary Hypothalamus Amygdala Hippocampus Pons mid-brain Cortex Pituitary Anterior pituitary

adult 80 days 80 days 29 days 80 days 80 days 80 days 42 days

Intermediate pituitary

14 days

pituitary

products

j3-Endorphin

-6, -2,6 and 25 days

-6 to 25 days

1 to 30 days

1 to 30 days

1 to 45 days -10 to 28 days, 3 and 9 months

1,7,14,21,28,35, 42 days and adult lo,29 and 80 days

N-acetyl B-endorphin

1,7,14,21,28,35, 42 days and adult

Bloom et all5

Tsang and Ng”’

Tsang et aIu9

Hompes et a1.53 Ng et a19i

Alessi et al.’

Kapcala65

Alessi et al. ’

Other endogenous opioids

Humoral endorphin

0, 1,2,3,4,5,6

Whole brain

3 weeks

Weissman et al.lz5

and 7 weeks a Determination of peptides as enkephalin equivalents. b Peak level determinations are quoted as maximal values and may in some instances be dependent on the age of testing.

urn-stimulated release of /?-endorphin lamic slices is low at 10 days and does pubertal levels until day 20 (ref. 53). Little work has been carried out in ficulties in obtaining material though of /3-endorphin have been made in a of cases. fi-Endorphin can be detected

from hypothanot reach postman due to difmeasurements limited number in the pituitary

at 8 weeks of gestation”. Levels of P-endorphin in plasma show a wide variation although increases are detected in both the mother and child at birthss. Further increases are seen in the cerebrospinal fluid of children if apnoea is present” which accords with studies in rabbits. The increased levels detected around birth remain high for approximately 4 days

402 and then fall to adult levels88. It has also been noted that the ratio of /?-endorphin to P-lipotropin is higher in neonates than in adults which like the studies in rats probably reflects differential processing of the

natal week in the cerebellum, the second postnatal week in the brainstem and at the third week in the forebrain, indicating a caudal-to-rostra1 sequence of development”’ although in contrast consistent in-

precursor peptide”‘.

creases in enkephalin

2.3. Proenkephalin products [Met]- and [Leu]-enkephalin

levels in all brain

regions

up

to postnatal day 25 have been reported13. A more detailed ontogenetic study of the opioid peptide have been detected

at similar time points to P-endorphin. Dahl et al.23 observed [Met]- and [LeuJ-enkephalin-like immunoreactive forms at embryonic day 13. However, HPLC separation revealed that the enkephalin measured was due to multiple immunoreactive forms. Of the [Leu]-enkephalin immunoreactivity, measured at embryonic day 16,70% was due to a form that eluted earlier than the [Leu]-enkephalin found in adult animals. This was decreased to 30% at 1 day and only 19% at 14 days postnatally. The multiple forms of [Met]-enkephalin were only detected prenatally. These multiple forms have also been detected in embryonic gut samples and limb buds and it has been suggested that they may be due to sulphated groups in the enkephalin molecule23. Others have not been able to detect enkephalin immunoreactivity until embryonic day 16 (refs. 13 and 15) and even then only at 0.08-2% of levels found in 25day-old animals. In addition, the distribution of enkephalin does not resemble adult patterns. Histological studies have detected enkephalin immunoreactivity at embryonic day 15 in the midline from the ventral pons to the cervical spinal cord94. In the lower brainstem”’ [Leu]-enkephalin immunoreactivity can be detected at embryonic day 16 whilst enkephalin-like immunoreactivity can be detected at embryonic day 18 in the central perikarya and processes98. In the neostriatum, however, [Metl-enkephalin activity is not detectable until embryonic day 20 (ref. 97). The hippocampal formation appears to develop rather later as [Leu]-enkephalin-like immunoreactivity is not detectable until postnatal day 4 (refs. 36, 37). In the North American opossum, which is born after only 12 days gestation and completes its development in its mothers pouch, enkephalin-like immunoreactivity is present at birth and increases to adult-like distribution patterns by 30 days2’. Peak levels of enkephalin determined by radioimmunoassay have been shown to occur in the first post-

products of proenkephalin

has been reported

by Bail-

ey and Kitchen6 within the striatum. A peak in [Met]enkephalin levels is observed at postnatal day 21 but the extended enkephalins [Met]-enkephalin-Arg6Phe’ and [Met]-enkephalin-Arg6-Gly’-Leu8 and [Leu]-enkephalin remain constant between 10 and 30 days. [Met]-enkephalin-Arg6-Gly’-Leu8 in hippocampal regions is not detectable at embryonic day 20 and shows a slight decrease from day 1 to day 7 but then increases rapidly by day 14 (ref. 135). As for /3-endorphin it is difficult to answer the question as to whether the proenkephalin system develops in parallel to its putative receptor type (the d-receptor). Binding studies with selective ligands agree that b-sites appear later in postnatal development and are fully developed by day 28 (Section 3.3.) but again there are interlaboratory differences in postnatal development. Nevertheless, where binding sites and peptides have been measured in parallel their ontogeny shows similarity96. Although the majority of work has been concentrated on the rat CNS, some studies have been carried out in the periphery. Adrenal [Met]-enkephalin content increases lo-fold from birth to adulthood while [Leu]-enkephalin increases only 4-fold79. The reverse is seen in the superior cervical ganglion where [Leu]-enkephalin increases lo-fold whilst [Met]-enkephalin increases 5-fold from birth to adulthood. Epstein et al.3”-32 have reported the ontogeny of [Met]-enkephalin in various areas of embryonic chick gut. It is first detected at 5 days of incubation in the rectum and duodenum. Levels in the rectum increase until 9 days of incubation and then remain constant whilst levels in the duodenum increase 4-fold by 13 days of incubation and then decrease to levels found in the 4-week-old chick. [Met]enkephalin is first detected in the cell bodies of the ganglion of Remak at 6 days. These cell bodies increase in number and produce processes into the gut wall by 9 days and varicosities of ganglion cells are present by 13 days of incubation. [Met]-enkephalin is

403 not detected

in the midgut until 9 days and increases

2-fold by 13 days of incubation. tected

[Met]-enkephalin

White et al.lz6 de-

in the ciliary

ganglion

of

stage 32 chick embryos. Levels increase 3-fold to a peak at stage 37 and then decrease until the adult levels are reached after hatching.

tor) has not been directly addressed togenetic

studies.

However,

in any of the on-

the rapid postnatal

de-

velopment with peaks in the peptide levels at the third or fourth week shows similarity with the receptor binding profile (Section 3.3.). 3. RECEPTOR ONTOGENY

2.4. Prodynorphinproducts Dynorphin and a-neoendorphin immunoreactivity in the neurointermediate lobe of the pituitary develop in parallel lo6. During the first 30 postnatal days both dynorphin

and a-neoendorphin

immunoreactiv-

3.1. Introduction Ligand binding studies have primarily been employed to follow opioid receptor development in foetal and neonatal animals. Binding studies using mem-

ity increase, with levels of dynorphin immunoreactivity being slightly higher. A more detailed study of the

brane fraction homogenates the main method employed,

products of prodynorphin shows that dynorphin-A, dynorphin,_s, dynorphin-B, a-neoendorphin and B-neoendorphin all progressively increase in the rat pituitary from birth to adulthood105. Studies of the molecular weight pattern of the prodynorphin products indicate that the neurointermediate lobes of the pituitary of neonatal rats have the ability to cleave two basic lysine-arginine residues of prodynorphin to produce dynorphin-A. However, the production of dynorphin,_s by cleavage at a single arginine residue from dynorphin-A is not as well developedlo5. This is evidenced by the concentrations of dynorphin,_, and dynorphin-A which are found at equimolar levels in the neurointermediate lobe of adults but in newborn rats the concentration of dynorphin-A is 3 times as high as dynorphin,_s. Also a-neoendorphin, which is converted to P-neoendorphin by cleavage of a lysine residue at the C terminal, is in equimo-

thalamic slices as an alternative has suggested that homogenisation reduces the number of viable binding sitest4. Binding studies in the adult have indicated at least 3 distinct types of opioid receptor @-, 6- and K-) and their ontogenetic profile has been examined. Since the early experiments of Coyle and Pert22 who used [3H]naloxone as a binding ligand the development of opioids more selective for the subtypes of opioid receptors has allowed a more detailed and accurate analysis of receptor ontogeny. [D-Ala*,Gly-ol]-enkephalin (DAGO) has been used as a selective ligand for the p-binding site. &Receptor binding has been demonstrated by using [D-Ala*,o-Leu’]-enkephalin (DADL) though suppression ofp-binding with an excess of DAGO is necessary because of the lack of selectivity of this ligand. More recently [D-kn2,DPen5]-enkephalin (DPDPE) which is highly selective

lar concentrations with /?-neoendorphin in adults but is 4 times more concentrated in the neurointermediate lobe of newborn rats. Thus it appears that processing enzymes involved in cleavage of single basic amino acid residues of these peptides are not fully developed at birth. Another study of prodynorphin ontogeny36 reported that dynorphin-A and /3-neoendorphin in the hippocampal formation develop later than seen in other CNS areas, levels not being detectable until postnatal day 6 and 7 respectively. This contrasts with dynorphin-A, dynorphin,_s, dynorphin-B and a-neoendorphin which are detectable at embryonic day 20 (ref. 135). The question of parallel development of prodynorphin products with its putative receptor (the K-recep-

for the 8-site21 has been used in our laboratory for studying the ontogenesis of 8-receptorss4. Opioids selective for x-binding sites have been slow to emerge and K-ligands such as ethylketocyclazocine and bremazocine bind also at the ,u- and o-sites and accordingly p- and d-binding must be suppressed with excess cold DAGO and DADL respectively. The benzodiazepine compound tifluadom has been shown to have selectivity for the Ic-receptorI and the K-selective Upjohn compound (U 69593) may prove to be a useful ligand in the future. Studies that have used less selective ligands without suppression of binding to other sites make data interpretation difficult 83.129.Although morphine and morphiceptin are relatively specific for p-binding sites both dihydromorphine and naloxone in addition

of brain tissue has been though the use of hypo-

404 TABLE II Ontogenesis of opioid binding sites in the rat CNS [3H]Binding ligand

Age

Brain region

Appearance of binding

Pea#

Increase in binding

Reference

[D-Ata’,D-Leu5]enkephalin

1,3,5,8,10,12,20 and 26 days 1,4,5,7,10,14, 21,28,35 adult

Whole brain

< 1 day

26 days

40 x

Wohltmann et al.lZ8

Forebrain

< 1 day

28 days

10 x

Spain et al.“2

10,14, adult lo,21 and 30 days

Forebrain

< 1 day

21 days

3x

Spain et al.“*

Whole brain

< 10 days

21 days

2.4 x

McDowell and Kitchens5

0,3,7,14,21 and 28 days, adult

Striatum

< 0 day

28 days

5x

Patey et al.96

Cerebral cortex


5x

0 to 28 days

Whole brain


Adult (>28 days) 28 days

2 x Low affinity 6 x High affinity

Zhong-Zhang and Pasternak136

[D-Pen’,D-Pen’]enkephalin

10,15,21,25,30,35, 40,45 and 50 days

Whole brain

25 days

4.5 x

McDowell and Kitchen84

Dihydromorphine

2 days and adult

Whole brain

< 2 days

Adult

2 x Low affinity 4 x High affinity

Zhong-Zhang and Pasternakt36

Diprenorphine

-8 to 30 days and adult

Spinal cord

-6 days

+ 6 days

30 x

Kirby68

Ethylketocyclazocine

14 and 21 days 2 and 14 days

Cortex Whole brain

< 14 days < 2 days

14 days 14 days

1,4,7,14,21,28 and 90 days 1,5,7,11,14,21 days and adult

Cerebral cortex

< 1 day

28 days

3 x High affinity 4x

Watanabe et al.‘24 Zhong-Zhang and Pasternakt36 Hill et a1.52

Forebrain

< 1 day

21 days

Forebrain

< 1 day

35 days

Hindbrain Whole brain

< 1 day < 1 day

4 days 16 days

2x 4 x High affinity 2 x Low affinity

Forebrain Brainstem Cerebellum Forebrain Brainstem Cerebellum Forebrain

< i < < < < <

1 day 1 day 2 days 1 day 1 day 2 days 2 days

23 days 14 days 4 days 22 days 14 days 4 days 24 days

12 x 5x 5x 13 x 5x 5x 18 x

Tsang and Ng’n’

Brainstem Cerebellum Forebrain Brainstem Cerebellum

< < < < <

2 days 2 days 1 day 1 day 2 days

14 days 5 days 22 days 14 days 4 days

7x 2x 10 x 5x 5x

Tsang et al.‘19

[D-Ala2,Gly-ol]enkephalin

[D-Ala’,Mets]enkephalinamide

1,4,5,7,

21,28,35

1,4,5,7,10,14, 21,28,35, adult 1,3,7,10,13, 16 days and adult

(Met]-enkephalin

1 to 30 days

1 to 30 days

2,5,11,14,24 and 30 days

1 to 30 days

10 days

2 x High affinity 4 x Low affinity 2x

Spain et al.“’

Spain et al.“*

Barr et al.”

Tsang and Ng”’

Tsang et al.“s

(continued)

405 TABLE II (continued) [3H]Binding ligand

Age

Brain region

Morphine

Naloxone

2 and 14 days

< 2 days

14 days

1.2 x Low

Increase

-

Reference

in binding

Pasternak et a1.95

2 and 14 days

Whole brain

< 2 days

14 days

1,5,10,15,20,25, 30,45 and 120 days 7,14 and 21 days

Brain minus cerebellum Spinal cord Medulla pons Midbrain Hypothalamus Striatum Cortex Whole brain

< 1 day

20 days

2x

< 7 days < 7 days < 7 days < 7 days < 7 days < 7 days -8 days

14 days 21 days 21 days 21 days 21 days 21 days 28 days

-7, -5, -2,o, 8, 13 and 28 days

Whole brain

-7 days

28 days

-7, -5, -2,o, 8, 13 and 28 days

Whole brain

-7 days

28 days

1.1 x 1.6 x 2x 3x 8.6 x 7.3 x 200 x (-8 Coyle and Pert’* to birth) 16 x (birth to 28 days) 25 x (-8 Garcin and Coyle3* to birth) 16 x (birth to 28 days) Garcin and Coyle39

1,5,10,20 and 30 days adult 1,2,5,10,14,20,25, 28 and 30 days

Brain minus cerebellum Forebrain

< 1 day

30 days

< 1 day

30 days

3x

Tsang et al.‘r9

Brainstem Cerebellum Whole brain

< 2 days < 1 day < 1 day

10 days 2 days 26 days

11 x 22 x

Wohltmann126

Spinal cord

< 1 day

6 days

Hypothalamic slices Whole brain minus cerebellum Brain minus cerebellum Whole brain

< 10 days

Whole brain

-9 days

1,3,5,8,10,12,20 and 26 days adult 1,4,6,8,10,15, 30 and 60 days lo,32 and 60 days 0, 1,2,3,4

and

5 weeks -7 to 140 days 2 days and adult

Phencyclidine

Peak”

affinity 3 x High affinity 1.2 x Low affinity 3 x High affinity

-8, -5, -2,o, 7,14, 28 days and adult

Nahrexone

Whole brain

Appearance of binding

-9, -7, -5, -3, -2, -1 and adult

a Peak binding determinations:

< 1 day

c -7 days

32 days 4 weeks

140 days

< 2 days

-1 day

Zhong-Zhang and Pasternak136

Auguy Valette et al5 Bardo et al.’

Koch et a1.76

Kirby and Mattio72 24% 16 x

Bhanot and Wilkinsonr4 Torda”’

8x

Clendeninn et al.‘O

3.7 x High affinity 1.6 x Low affinity

Zhong-Zhang and Pasternak’%

10 x

Simar and Zukin”’

these are quoted as maximal values and may in some instances be dependent on the age of testing.

bind at both 6- and Ic-sites@, whilst [Met]-enkephalin, [Leu]-enkephalin and [D-Ala*,Met]-enkephalinamide bind at both the 6- and,u-sites with similar affi-

nities. Studies with these ligands have revealed little about the development of the receptor subtypes. Naltrexone, diprenorphine and etorphine bind to all

406 3 types of receptor

and therefore

studies using such

ligands can only indicate total opioid receptor binding. Comparison of data is hampered by the variety of methods

used to express results.

For example

with

postnatal week. Some disagreement since Torda115 found peak naloxone

appears binding

here at 1

month in rats. The regional distribution of binding is dependent on the age of the animal. Naloxone binding studiesX

respect to maximal binding capacity data may be represented per brain, per milligram wet weight or per

have shown marked

milligram protein. This may produce apparent differences in the time of peak levels and anomalies in

ing between the newborn and adult have been confirmed in other laboratories38~3g~1’5~11’and for other

rates of receptor increase. As was seen with endogenous study of opioid receptor ontogeny

ligands such as [Met]-enkephalin116-1’y opioid levels the has primarily been

carried out on the rat CNS (Table II). Prenatal receptor ontogeny studies have used only whole brain homogenates due to problems associated with size of foetal brain regions, dissection and accumulation of sufficient material. Only a few groups have carried out complete saturation curves in postnatal studies, and most studies have employed a single concentration of ligand to measure binding to receptors. This limits the amount of information that can be obtained as no measure of affinity or receptor concentration can be made. Those that have carried out saturation curves are in general agreement that increases in binding with age are due to increases in the concentration of receptors (B,,,) and not the affinity of the receptor for the ligand

cKd)

11,14.20.22,38,41.84,85

3.2. Receptor

ontogeny

in the rat: non-selective

li-

gands

Binding of naloxone in rat wholebrain can be detected as early as embryonic day 15 (refs. 22,38) and in the spinal cord binding of diprenorphine can be shown at embryonic day 16 (ref. 68). Using histological techniques Kent et al.67 first detected naloxone binding in the striatum between embryonic day 12 and 14 and at embryonic day 16 in the paleocortical olfactory areas and the medial septum. Enkephalin binding could not be detected until embryonic day 20 in the striatum and these findings correlate with the first appearance of endogenous opioid peptides as described in Section 2. As was seen with most of the opioid peptides, receptor binding increases with age to a peak during the third postnatal week except for the cerebellum which shows a peak in [Met]-enkephalin binding 116-11gand naloxone bindingllg during the first

and 21 days. Regional

morphiney9. Autoradiographic [3H]etorphine121,

regional

differences

differences

studies

between

in naloxone

7

bind-

and dihydro-

with [3H]naloxone8h.y’,

[1251]DAG087 describe

the forma-

tion of clusters of opioid receptors as patches which appear in striatal regions in the first postnatal week and persist into adulthood as opposed to the diffuse labelling seen from prenatal day 14 (ref. 87). These patches are in topographical register to the patches of dopamine fluorescence that are seen in the striatum from prenatal day 19 to postnatal day 16. Patches of opioid receptors are also observed in the caudate-putamen at postnatal day 5 (ref. 121). Naloxone binding has been detected in the nucleus accumbens in the form of patches from prenatal day 18 but in contrast this is not in close association with dopamine fluorescence patchess7. Autoradiographic studies with dihydromorphine show binding in the hippocampus and olfactory bulb at postnatal day 2 (ref. 120). Maximal densities of dihydromorphine binding are detected at day 14 in the caudate, hippocampus, amygdala and hypothalamus but are not obtained until day 21 in the medial thalamus and quadrigeminal plate. Highest levels of binding are detected in the interpeduncular nucleus followed by the habenula, hypothalamus and periaqueductal greyYy. Sex differences have also been shown in studies of the autoradiographic localization of opioid receptors using [3H]naloxone. In the medial preoptic area there is an increase in opioid receptor density from day 3 to day 10 in the female which is absent in males4s. 3.3. Receptor ontogeny in the rat: selective ligands The use of relatively selective ligands has allowed the differential development of opioid receptor subtypes to be studied. Koch et al.76 demonstrated that the low affinity binding site for naloxone was not present at postnatal day 1 but at subsequent ages both high and low affinity sites are present. DADL

407 binding

has been

compared

with dihydromorphine

and with naloxones’*‘28. Leslie et al.‘l showed stereo-

ential ontogeny. Peak binding for DAGO at day 21 has been confirmed by other workerss5 as has the lat-

dihydromorphine and DADL the ,u-ligand morphiceptin inin the neonate but not in the that in the neonate both di-

er development of &receptors using the highly selective ligand [D-Pen2,D-Pen5]-enkephalin84. With this

hydromorphine and DADL bind to p-receptors. In older animals as morphiceptin no longer inhibits DADL this probably reflects binding to &receptors. Wohltmann et al.‘*s obtained similar results using

is more disparity in the literature for the x-receptor. Peak binding being reported as late as day 35 (ref. 112) and in a most recent study as early as day 16 (ref. 12) using ethylketocyclazocine as a binding ligand.

DADL as a ligand. DADL binding was lower than naloxone binding but increased at a parallel rate till

There are indications

specific binding to both at 6 days and adult, but hibited DADL binding adult. Thus it appears

day 12. Thereafter, DADL binding increased at twice the rate of naloxone binding until day 26 when the adult 1:l ratio was obtained. Naloxone binding could be completely displaced by morphine in neonates and adults. In contrast DADL was only completely displaced by morphine up to postnatal day 12. At later ages only partial displacement was observed leaving some specific residual binding of DADL. These results suggest that DADL and naloxone bind to a similar site during the first two postnatal weeks but then DADL binds to two different sites, probably p- and CC. Hill et a1.52 using ethylketocyclazocine showed specific binding from day 1 increasing to adult levels by 28 days. Tifluadom, a specific K-receptor agonist, was 3 times as effective at inhibiting ethylketocyclazocine binding to the cortex of neonates than in adults indicating that in the neonate there is a higher ratio of K- to p-receptors in the cortex than in the adult. The p-receptors must therefore increase more rapidly during development to reach adult ratios of rcto ,u-receptors where tifluadom would be less potent in inhibiting ethylketocyclazocine binding. A comparative study of the ontogeny of p-, 6- and x-receptors has been reported by Spain et al.“* using DAGO to follow p- and DADL and ethylketocyclazocine with suppressor ligands to follow 6- and rc- respectively. DAGO binding was found to decline for several days after birth but then increased two-fold over the subsequent two weeks to reach adult levels. d-Receptor binding did not appear until the second postnatal week and increased 3-fold between postnatal day 10 and 28. K-Receptor ontogeny using ethylketocyclazocine was low at birth and had only increased two-fold by the fifth postnatal week. It is thus clear that the receptor subtypes exhibit a differ-

ligand b-receptors cannot be detected before postnatal day 10; and peak binding is seen at day 25. There

that this ligand binds to more

than one site in the neonate”* even where experimental measures are taken to suppressp- and d-binding and this may contribute served between laboratories.

to the differences

ob-

3.4. Receptor ontogeny in other species Several groups have studied opioid receptor ontogeny in species other than the rat. The ontogeny of opioid receptors in chick has been reported by Gibson and Vernadakis41 and Bardo et al.“. Gibson and Vemadakis detected etorphine binding in chick embryos as early as 4 days incubation. By day 10 of incubation binding activity was confined to neural tissue. Scatchard analysis showed the increase in binding to be due to increases in receptor concentration and not changes in receptor affinity. Bardo et al.‘l followed post-hatch development of naloxone binding in the midbrain and forebrain of the chick and found numbers of binding sites increased in both areas but to a greater extent in the forebrain. However, the density of receptor sites showed decreases in both areas and this was more marked in the midbrain. Again, there were no accompanying changes in receptor affinity. DAGO binding in the mouse increases markedly between 3 and 15 days and shows further increases when measured at 10 weeks114. b-Binding using DADL with p-binding suppressed could not be detected at 3 days and showed increases in sites at day 15 and at 10 weeks of testing. This accords with the later development of &sites in the rat. K-Binding using bremazocine with ,u- and b-binding suppressed was detectable at 3 days and increased with age. The regional distribution of naloxone and [D-Ala*, Met5]-enkephalinamide binding in the lamb has been described’22. Highest levels of naloxone and [D-Ala*, Mets]-enkephalinamide binding are found in the striatum and hypothalamus followed by the mid-

408 brain,

thalamus,

temporal

cortex,

occipital

cortex,

pons, medulla and hippocampus. All areas show an increase in binding from 68 days gestation to birth at 147 days gestation, except for the pars medulla where a decrease in binding is observed. After birth binding plateaus or decreases slightly to adult levels. In sheep ,u- and &receptor binding has been shown in foetal and maternal brain regions. In the frontal cortex and hippocampus dihydromorphine binding to p-receptors is higher in foetal sheep brains at 118127 days gestation’s. This difference, however, is not observed in the cerebellum, a region rich in dihydromorphine

binding

sites at all ages. &Receptor

binding demonstrated by DADL binding also shows peak levels in foetal brains at 118-127 days gestation”s. The studies in this species demonstrate a more fully mature receptor system at birth than is observed in rats and mice. 3.5. Peripheral receptor ontogeny Peripheral opioid receptor development has been reported by Gintzler et al. 43. Diprenorphine binding in the enteric nervous system of the guinea pig was detected at 25 days gestation. Concentration of receptors remained fairly constant between 25 and 50 days gestation except for a transient fall at 30 days. The amounts of ligand bound increased throughout the period, the transient fall being due to rapid increases in protein concentration.

4. PHARMACOLOGY

OF THE OPIOID SYSTEM DUR-

ING ONTOGENY

4.1. Introduction Much of the pharmacology of morphine and its analogues during development preceded work on receptors and on levels of endogenous opioids. As early as 1911 the increased sensitivity of neonatal rabbits to morphine had been reported2’ and in 1938 similar effects in rats were shown4’. Since these early reports many studies have been carried out on various aspects of opioid pharmacology in the neonate. These studies primarily in the rat include antinociception, pharmacokinetics and toxicity (Table III). Some work on isolated tissue responses to opioids, taken from neonatal animals, has also been reported.

4.2. Antinociception Several antinociceptive tests have been used including tail flick4,47,95,13h,tail immersion5~‘2~75, hot plates’.6i,62 , paw pressure74, ultrasonic pain stimulation93, electric algic stimulation”’

and in the guinea

pig dental pulp stimulation34. The majority of studies have used morphine as the antinociceptive drug, though effects of phenoperidine, pethidine and ketocyclazocine have also been reported5g74q92. The peak antinociceptive effect usually appears during the second or third postnatal week. Differences have been reported in the ontogenetic profile of the antinociceptive effects of morphine and ketocyclazocine’2 where there are indications

that ketocyclazocine

an-

tinociception is observed about 4 days earlier than for morphine raising the possibility that functional Ksites are operative before p-sites. It has also been reported that tolerance to analgesic effects of morphine is present in neonates57.92 and that peak tolerance occurs at 21 days. Two groups have reported the development of nociceptive responses of neonatal rats. Hamm and Knisely4’ used the tail flick assay and found no difference in nociceptive threshold in lo-day, 28-day and 5-7-month-old rats. Kitchen and McDowell74 using the paw pressure test found that with increasing age (lo-30 days) a greater pressure was required to elicit a nociceptive response. The analgesic action of morphine in guinea pigs3” shows it to be less effective against dental pulp stimulation in 4-5-week-old animals than in lo-week-old and adult guinea pigs. Thus although CNS maturity in this species appears to be more advanced than in the rat and rabbit, as evidenced by walking and feeding behaviour, opioid system function lags behind by several weeks. 4.3. Locomotor activity The effect of opioids on locomotor activity in the developing neonate is contradictory with both hypoand hyperactivity being reported. Using activity meters, methadone produces hypoactivity in animals less than 25 days of age and this is prolonged in neonates 12 days old or less lo8 However, in rats older than 20 days post-partum morphine administered intraperitoneally produces hyperactivity which is most marked at later ages cd5 . Using line crossing as a measure of activity, lo-day-old neonates exhibited hy-

409 poactivity methadone

after morphine

administrationus.

later ages exhibit

a different

As for profile.

In

17-day-old neonates, low doses of morphine (0.5 mg/kg) produce hyperactivity, but doses of 5 mg/kg produce hypoactivity and by 24 days no significant effects on activity were observed at any dose18. In mice, 1 mg/kg morphine

has no effect on activity

except at 20-22 days where activity is depressed. After 10 mg/kg mice show hyperactivity except at 20-22 days when hypoactivity is observed33. Thus in both neonatal rats and mice, it appears that age of testing and dose of opioid used are important determinants of the activity changes. 4.4. Opioidpharmacokinetics The variations observed with dose and age on locomotor activity and analgesia may be partly due to changes in distribution and metabolism of opioid drugs during the neonatal period. Until day 14 the blood-brain barrier does not begin to form24 and therefore levels of these drugs entering the brain are higher than in older animals. Several workers have reported that blood-brain ratios of opioids increase from day 14 to a maximum at approximately 30 days ‘9” though peak brain and tissue levels of Lmethadone occur at 8 days post-partum’08~‘w. The metabolic activity of the neonate also varies with age. Glucuronidation of morphine reaches a peak by day 7 (ref. 130) while 0-demethylation, Ndemethylation and esterase activity all reach their peak activity at 35 days post-partum but are first detected at different times13’. 0-Demethylation is the first to appear at 3 days, N-demethylation appears at 7 days but esterase activity does not appear until 14 days post-partum. Other workers have also shown the conjugation of morphine to be increased with age”. In 16-day-old rats only 9% of the morphine dose is conjugated within 45 min whereas in 32-dayold rats 55% of the dose appears in the conjugated form. There are no reports of the ontogeny of metabolic enzymes for the endogenous peptides but enzymes involved in processing of the precursor molecules exhibit differential postnatal development (Sections 2.2.-2.4.). 4.5. Toxicity Changes in metabolism

and distribution

are also

reflected

in the toxicity of opioid drugs in rats. Mor-

phine-induced catalepsy reaches a peak at 20 day@ whereas the hypothermic effect of methadone peaks at 8 days post-partum lo8. The LD,, of morphine is relatively constant at 50 mg/kg during the first two weeks after birth but then at 16 days the LDsO value rises to adult levels of 220 mg/kg”. This may be due to a combination of the development of the bloodbrain barrier from day 14 and the appearance of the various metabolising systems which appear mainly during the second postnatal week, but may also be related to the ontogeny of receptor subtypes which do not develop in parallel”* (see Section 3.3.). Rabbits have also been used to study the neonatal toxicity of morphine. Eddy29 and Schlossman’04 reported that the lethal dose of morphine increased from birth until 2-3 months indicating an increase in metabolising enzymes and a decrease in the sensitivity to morphine. Eddy29 also reported that the dose causing convulsions was close to the lethal dose in young rabbits but became lower than the lethal dose in older animals. 4.6. Respiration and cardiovascular system The involvement of the opioid system in ontogenesis of respiratory control has been investigated. In rats the respiratory depressant effects of morphine, [D-Ala*,Met’]-enkephalinamide or ,&endorphin are similar between 2 and 14 days of age95*136.However, in rabbits alterations in opioid function show abnormal effects on respiration in the neonate. Hazinski et al.51 demonstrated that infusions of naloxone increase respiration by 140-180% in the majority of pups less than 4 days old while in pups greater than 5 days old this response is only observed in a minority of animals. Naloxone administered during hypoxia also stimulates respiration but this response decreases with age 46. In addition the administration of DADL decreases ventilation in rabbit pups and this effect is inversely proportional to age45. Naloxone was also shown to increase phrenic nerve minute output (a neural index of respiratory output) by 122% in piglets less than 10 days old but only increased output by 54% in piglets aged 20-34 days’*. Thus it appears that respiratory control in neonates is partly under the control of the opioid system but that this effect diminishes with age and is species dependent. The effect of [Met]-enkephalin on the circulatory

410 TABLE

III

Ontogenesis of opioid pharmacology in the rat _ Pharmacological Age test _~~_. _ -Antinociception Tail immersion 5,10, 15,20,25,30,45, 60 and 120 days

Drugs used

Effect

Morphine

Peak antinociception at day 15 decreasing to day 45 Peak antinociception days 15 to 25 decreasing to day 45 Peak antinociception at day 14 Peak antinociception at day 10 No effect of age on nociceptive responses Foot shock induced analgesia increases to day 28 Peak antinociception at day 12 to 13 Peak antinociception at day 20 decreasing to day 42 Decrease in antinociception with age Peak antinociception at day 14 decreasing to day 32 then, increasing to day 120 Greater antinociception at day 14 Peak antinociception at day 14

Phenoperidine 3.5,7,9,10,12,

14 days

Morphine Ketocyclazocine

Tail flick

lo,28 days and 5 to 7 months

Hot plate

12 to 13 days and 20 to 21 days 20.26,32 and 42 days, adult 16,25 and 38 days

Morphine

Ultrasonic pain stimulation

14,17,30,60,90 and 120 days

Morphine

Tail flick

2 and 14 days 2.3,7,11 and 14 days

Morphine Morphine

Electric algic stimulation

21 and 42 days, 3 and 18 months

Morphine

Morphine Morphine

Pethidine

Catalepsy

20,50,80

Distribution

Isolated tissues Vas deferens

Duodenum

Maximum tolerance to antinociceptive effects at day 21 decreasing to 18 months Maximum tolerance to antinociceptive effects at day 21 decreasing to 18 months

Auguy-Valette

Barr et al.” Hamm

and Knisely”

Johannesson and BeckerhI Johannesson and Becke@* Huidobro and Huidobro5’ Nicak and Masnyk’j

Pasternak et al.’ Zhong-Zhang and Pasternakt3’ Nicak and Kohut’l

Morphine

Peak catalepsy at day 20 decreasing to day 110

Katz”’

5,10,15,20,25 and 30 days 16 and 32 days

Morphine

Blood brain ratio increases from day 14 to day 30 Blood brain ratio greater at day 32

Auguy-Valette

1,4,8,12,20 and 60 days 1,8, 12 and 20 days

Methadone

10,20,30,40,50,60, 90 and 120 days

Morphine

Peak IC,, at day 60

/3-Endorphin [Met]-enkephalin

No difference in IC,,, Peak relaxation at day 18. first detected at day 8 Peak relaxation at day 18. first detected at day 8

and 110 days

1.3,8,10,15,18,20,25, 30,35,40 and 45 days

Morphine

Methadone

Morphine

Locomotor activity lo,17

and 24 days adult

1,4,8, 12,20and 25 days 20,50,80 and 110 days

Morphine

Methadone Morphine

Peak brain levels and tissue accumulation at day 8 Peak brain synaptosomal levels at day 20

Hypoactivity at day 10. hyperactivity and hypoactivity at day 17 and adult Greater hypoactivity in 1. 4 and 8 day old Hyperactivity increasing to day 80

et al.”

et al.’

Kupferberg and Leong-Way” Shah and Donalda’s Shah et al.“”

Huidobro-Toro Miranda’” Furukawa

and

et al.ji

Caza and Spear’#

Shah and Donald”” Katzhh (conrinued)

411 TABLE III (contiflued) P~a~aco~ogicaZ test Metabolbm

Age -

Drugs used

EJfect

Reference

Morphine

Peak elevation of striatal DOPAC at day 15 Peak glucuronidation at day 7

Roth et al. lo1

3,6,10,15,19

and

35 days 1,3,7,10,14,21,28 and 35 days adult

Morphine Norcodeine Meperidine

Plasma cyclic nucleotide level

1,3,5,7,9,14,21,

Respiratory depression

2 and 14 days 2 and 14 days

Temperature

Morphine

28and49days

1,4,8,12,20

Peak ~-demethylation at day 35 appears at day 1 Peak N-demethylation at day 35 appears at day 3 Peak esterase activity at day 35 appears at day 7 Peak eievation of CAMP and cGMP at 21 days decreasing to day 28

No difference depression No difference Morphine depression ID-AIa”,Met’]No difference enkepha~inam~de depression No difference /3-Endorphin depression Morphine

Yeh and KrebslM

Muraki et aLa

in respiratory

Pasternak et aL9’

in respiratory

Zhong-Zhang and Pasternakt3”

in respiratory in respiratory

Methadone

Peak hypothermia at days 1, 4and8

Shah and Donaldto*

and 25 days Toxicity

1,2,4,8,12,16,24 and 32 days

Morphine

LD, increases after day 16 to day 32

Kupferberg and Leong-Way”

Food intake

3, 10,12,14 and

Naloxone

No effect of naloxone suppressing food intake until day 14

Aroyewun and Barr’

19 days

responses in sheep have been reported7’ in foetal and newborn lambs, and in pregnant and non-pregnant ewes. [Met]-Enkephalin administered i.v. causes decreased heart rate and blood pressure in foetal and neonatal lambs. In contrast in pregnant ewes, however, this is preceded by an initial increase in heart rate and blood pressure. 4.7. Isolated tissues In the rat vas deferens age does not affect the potency of ~-endo~hin to inhibit field-stimulated muscle contraction5*. However, the potentiating effect of morphine in this tissue, which is not naloxone sensitive, increases with age to a maximum at 2 months. Rat duodenum responses to [Met]-enkephalin alter during development35. From day 8 there is a sustained relaxation of the tissue to [Met]-enkephaIin reaching a maximum at 18 days. This response can be blocked by tetrodotoxin or naloxone but by day 40 it

is no longer tetrodotoxin sensitive. This indicates a change from neurogenic to myogenic activity of opioids during development of the gut.

4.8. Genotype var~t~o~ Abnormal development of peptide levels has been found in obese mice of the C57 BL/6J oblob strainlDO. Hyperphagia accompanied by increased weight occurs during the first 3 months of life. &Endorphin levels in the pituitary are found to be increased above normal from 3 months and would therefore appear to be a consequence rather than cause. However, posterior pituitary levels of enkephalin show a two-fold increase from 1 month to 6 months and correlate well with changes in body weightIN. Rats of the Naples Sprague-Dawley derived high excitable form are more sensitive to injection of [Leu]- and [Met]-enkephalin into the neocortex than the low excitable form as indexed by electrophysio-

412 logical measurements.

Sensitivity

to this effect in-

creases with age”*. 4.9. Miscellaneous

ceptive response to morphine in lZday-old pups. However, Steele and Johannesson’i3 who used a similar regime found that pups aged 20-32 days from

The ontogeny of opioid effects on growth hormone has been reported for the ovine foetus44, P-endorphin

morphine-injected dams showed no difference in morphine antinociception. This tolerance to morphinell was suggested to be due to a suppression of

increases the secretion of growth hormone but the magnitude of the response varies inversely with age. Opioids stimulate plasma cyclic AMP and GMPg9. In the neonate morphine or the p-agonist FK 33-824

cell division Morphine administration to the dam during pregnancy has also been reported to cause a transient increase in [3H][Met]-enkephalin binding during the

does not increase cyclic AMP until 3 days post-parturn and does not increase cyclic GMP until 7 days post-partum. This reflects the immaturity of the opi-

first week post-partum but drops below normal levels in the following 3 weeks in the brainstem and forebrain returning to normal in the cerebellum116. Iyengar and Rabn“s9 also reported increased binding

oid system and not plasma cyclic nucleotide function since noradrenaline and carbachol are capable of stimulating plasma cyclic nucleotides in l-day-old rats. Peak effects of opioids upon cyclic nucleotides are seen at 21 days which correlates well with both receptor and opioid level ontogenyg9. The ability of morphine to alter the metabolism of dopamine has been monitored during development”‘. Morphine-induced elevations of DOPAC are detected in the striatum at day 6 post-partum and day 3 in the adrenal medulla. The maximum effect in both areas is seen at 15-19 days”‘. 5. MODULATION

OF THE ONTOGENY

OF THE OPI-

OID SYSTEM

5.1. Introduction In common with the development of other neurochemical systems opioid ontogenesis has been shown to be sensitive to perinatal exposure to drugs, insecticides and heavy metals (Table IV). The picture obtained from such studies is complicated due to variations in exposure time, dose and route of administration, the test used to detect any modulation of development and the ages at which pups are tested. All vary from study to study which inevitably makes comparison difficult. 5.2. Opioid administration The problem of varying experimental protocol can be illustrated by the work of Johannesson and Becker61 who found that prenatal injection of morphine to the dam produced tolerance to morphine antinociception in lZday-old pups whereas infusion of morphine to the dam63 actually increased the antinoci-

in 5-,30- and 35day-old pups from morphine-treated dams whereas in contrast decreased binding at birth but not in 60-day-old pups from morphine-treated dams has been shown69. Morphine administration to neonates early in life also increases p-binding in the striatum and nucleus accumbens, with no change in d-receptor function49. However, in balance other studies have failed to find changes in binding in morphine-treated animals. Bardo et al.9 could find no effect on naloxone binding in 22-day-old pups after 21 days of morphine treatment although tolerance to morphine was reported. This has been confirmed by another study where morphine and naloxone were administered in the last week of pregnancy2*. In the chick, LAAM (L-a-acetylmethadol) was found to effect [3H]etorphine binding in embryonic brain; B,,, was decreased while K, was increased in all ages of chick embryo used42. With respect to antinociceptive responses, 25- and 120-day-old rats from morphineor methadonetreated dams show a decreased response latency in the tail flick test” though methadone administration to the dam has also been shown to increase latency to nociceptive stimuli in rat pup~‘~‘.‘~*. An increased threshold to painful stimuli has also been reported for 2- to 7-day-old pups administered /3-endorphin”“. Aroyewun and Barr found no change in naloxoneinduced anorexia in pups from morphine-treated dams but when morphine was administered to the pups for the first 5 days post-partum the naloxone-induced anorexia was detected several days earlier than normally seen with control animals3. Long term receptor blockade during postnatal development has been studied by administration of na-

413 TABLE

IV

Modulation of the ontogeny of the opioid system in the rat Age of testing

Effect on development

Reference

90 days

Increased nociceptive threshold

Sandman et al.‘“’

[3H]Nd~~~ne binding

-1 and 24 days

Decreased B,,, in spinal cord at -1 day. No effect 24 days Increased Kd in brainstem at -1 day. No effect 24 days

Kirby et al.”

Pituitary [Met]enkephalin Pituitaryb-endorphin Hypothalamic [Met]enkephalin Hypothalamic fl-endorphin

21,70,120 and 180 days

Decreased levels in males at 70 and 120 days No effect No effect

Hong and Ali”

Adult

Increase in sensitivity to morphine analgesia

Larsonso

14 and 21 days

Decreased binding in cortex and striatum at day 14

Watanabe et a1.‘24

Age of treatment

Test

/%Endorphin

2 to 7 days

Antinociception tail flick

Capsaicin

-7 and -6 days

4 days

Modulating system

Chlordecone (Kepone)

Schedule of treatment

1mgipw

-

Chronic stress

Saline s.c. twice daily

5 to 33 days

Morphine

Diazepam

20 mg/kg/day

Dam from con. ception and pups day 1 to day 10 Pups day 1 to day 10

[3H]Ethylketocyclazocine binding

0 and 2 days

[3H]NaIoxone

binding

1,4,6,8, 10, 15,30 and 60 days

Decreased binding by 22% at day 6 and 32

Kirby and Mattio”

1 to 21 days

[‘H]Naloxone

binding

7.14 and 21 days

No effect

Bardo et al.’

Activity, nociception, temperatures Activity, nociception, temp. after morphine

21 days

Increased nociception in footshock treated animals Decreased effect of morphine in footshock treated animals

-21 to 0 days

[‘HlNaloxone

0 and 16 days

Decreased binding greater at 0 day

Moon@

5,7-Dihydroxytryptamine Footshock

30 m&day of 1 shock/30 s, lmA,ts

analgesia

Decreased levels in rats of both sexes at 21 days

No effect

Haloperidol

7 mg/week or 12 mg/3 week osmotic mini

Lead

0,300 and 1000 ppm PbAc in maternal drinking water 0,100,300 and loo0 ppm PbAc in maternal drinking water

-21 to 21 days

Striatal enkephalin levels

10,21,30, 40,50 and 100 days

Decreased levels at 10 and 21 days

Winder et al.“’

-21 to 21 days

Striatal (Metl-enkephalin levels

lo,21 and 30 days

Decreased levels at all ages and doses of lead

Bailey and Kitchen”

0,300 and loo0 ppm PbAc in maternal drinking water 0,300 and 1000 ppm PbAc in maternal drinking water

-21 to 21 days

binding

pump

-21 to 21 days

Striatal [Metl-enkephalyl-Arg6-Phe’ Striatal [Metl-enkephalyl-A&-Gly’-Leus Striatal [Leu]enkephalin Morphine antinociception - tail immersion

Ketocyclazocine antinociception paw pressure

-

Decreased levels at 10 and 21 days Decreased levels at 10 and 21 days Decreased levels at all ages lo,21 and 30 days

Decreased antinociception at day 10

Kitchen et a1.75

lo,21 and 30 days

Decreased antinociception at day 10 for 300 ppm and at day 10 and 21 for loo0 ppm

Kitchen and McDowell74

(continued)

414 TABLE

IV (continued)

Modulating system

Methadone

Schedule of treatment

Age of treatment

Test

Age of testing

Effect on development

Reference

0,100.3OOand loo0 ppm PbAc in maternal drinking water

-21 to 14 days

[3H]DAG0 binding

lo,21 and 30 days

No effect

McDowell and Kitchen*’

5 mg/kg/day 1.p. to dam

5 days prior to mating to birth 5 days prior to mating to weaning 0 to 21 days

Nociception hot plate 66 “C

21.45,60,120 and 300 days

Zagon and McLaughlin”’

S mgikgiday i.p. to dam

5 days prior to mating to birth 5 days prior to mating to weaning

Nociception hot plate 55 “C

21,30,45, 60,90 and 120 days

Increased nociceptive threshold at 21,45 and 120 days Increased nociceptive threshold at all ages peak at 60 days Increased nociceptive threshold at 45,64l and 120 days Increased nociceptive threshold at 21,30,45 and 60 days Increased nociceptive threshold at 21,30,45 and 60 days. Females also at 90 and 120 days Increased nociceptive threshold at 21,30.45, 60 and 90 days Decreased antinociception at both ages Decreased antinociception at both ages Increased binding in striatum and nucleus accumbens No effect

Handelmann and Quirion”’

0 to 21 days

Morphine

5 mgikgiday i.p. to dam

-28 to 21 days

Antinociception hot plate 55 “C Antinociception tail flick

25 and 120 days

1pegs.c.iday

1 to 7 days

[‘H]Naloxone binding

6 months

[JH][D-Ala2.D-Leu’]-

20 mgikgiday s.c to dam 5 mgikgih for 4 h i.v. to dam 20 mglkgiday s.c to dam

-5, -4,-3 and -2 days -1 day

5 mgikgih for 4 h i.v. to dam 10 mgikg i.p. twice daily to dam

-1 day

-5,-4,-3and -2 days

-28 to 0 days

7.5 mgikg S.C. twice daily to dam 5 mgikgiday

E5 to 0 days

5 mgikgiday 1.p. to dam

-28 to 21 days

5 mgikg twice daily

1 to 21 days

enkephalin binding Antinociception hot plate 55 “C Antinociception hot plate 55 “C Antinociception hot plate 55 “C Antinociceptionhot plate 55 “C [3H]Met-enkephalin binding

Naloxone-induced anorexia

12 and 21 days

Decreased antinociception with morphine 12 and 21 days Increased antinociception with morphine 12 and 21 days Decreased duration of 26 and 32 days antinociception 12 days

0 to 30 days

lo,12 and 14 days

1 to 5 days

Antinociceptionhot plate 55 “C Antinociception tail flick [‘H]Naloxone binding Antmociception

25 and 120 days

22 days

Increased antinociception with morphine Increased binding from 0 to 7 days then decreased binding in forebrain Increased binding from 5 to 7 days otherwise binding greatly depressed in brainstem Increased binding especially at day 5 in cerebellum No effect of pretreatment

Suppression of food intake in younger animals than normal Decreased antinociception only significant at 120 days No significant decrease in antinociception No effect

Zagon and McLaughlin’32

Hovious and Peters’s

Johannesson and Becker” Johannesson et al.63 Steele and JohannessonIl

Tsang and Ng”’

Aroyewun and Barr’

Hovious and Peters”

Bardo et al.’

Tolerance to morphine antinociception (continued)

415 TABLE IV (continued) Modulating system

Naloxone

Schedule of treatment

Test

Ageof

10,15 or 20 mglkglday to dam 20 mg/kg/day in 4 doses 20 mglkgiday in 2 doses 20 mglkgiday in 4 doses

-17 to-10 days

5 mg/kg i.p. twice daily to dam

-28 to 0

Effect on development

Reference

Tolerance

Morphine-induced hypoactivity Opiate receptor binding

Iyengar and Rabii59

-1Oto-l

days

Morphine analgesia

5,10,15,20, 25,30 and 35 days 30 days

-1Oto-l

days

Morphine analgesia

30 days

Increased receptor numbers at 5,30 and 35 days in morphine-treated group Increased analgesia - earlier onset, longer duration No effect

-10 to-l

days

[‘H]Naloxone binding

-1 and 60 days

Decreased B,,,

Kirby and Aronstam@

[‘H][Met]-enkephalin binding

0 to 30 days

No effect in forebrain

Tsang and Ng”’

1 mgikg twice daily

1 to 21 days

[‘H]Naloxone binding

22,24,28 and 35 days

1 mg/kg twice daily

1 to 21 days

[‘H]Naloxone binding

22 days

0.5 mgikg to dam

E20

Morphine analgesia Morphine-induced hypoactivity Antinociceptiontail flick Tail flick nociception

20 mg/kg day 3 to 14 and

3 to 20 days

23-h Deprivation study

2 to 7 days

Naltrexone

Age of testing

treatment

90 days

120 days

24-h Fluid intake study

60 mgikg day 15 to 20 10 mglkglday

1 to 5 days

Naloxone-induced anorexia

lo,12 and 14 days

1,10,20or50 mglkglday

1 to 21 days

Morphine analgesia hot plate

22 days

-

loxone or naltrexone to the pups during the first few weeks after birth. This results in increased binding in the hypothalamus, striatum and cortex after 21 days to the exposure lo. Indeed, naloxone administration dam produces a transient increase in [Metl-enkephalin binding in the brainstem116. In addition increased nociceptive latency in hot plate tests in 90-day-old pups exposed to naloxone from day 2 to 7 has been observed” and decreases in morphine analgesia after 21 days exposure to naltrexone134. Pups exposed to naltrexone from 3 to 14 days post-partum show a de-

-

Increased binding 0 to 8 days then greatly depressed in brainstem No effect in cerebellum Peak increase in spinal cord, hypothalamus, striatum and cortex at 21 days Increased B,,, in spinal cord, hypothalamus, striatum and cortex Decreased K,, in spinal cord and cortex No effect No effect Increased nociceptive threshold Decreased pain threshold Decreased naloxone suppression of milk intake Decreased naloxone suppression fluid intake Effect of naloxone abolished at 10 and 12 days restored at day 14 Decreased for 6 h with 1 and 10 mg/kg, 24 h with 20 and 50 mg/kg

Kirby et al.‘”

Bardo et al.‘”

Bardo et al9

Auditore et aLJ Diaz et aLi’

Aroyewun and Barr Zagon and McLaughlin’34

crease in the naloxone-induced anorexia whereas morphine administered from 1 to 5 days post-partum increases naloxone-induced anorexia25. The behavioural effects of perinatal opioid exposure are known to cause withdrawal, delays in behavioural maturation, impaired cognition, etc., and these effects have recently been reviewed by Zagon and McLaughlin’33. 5.3. Stress The effect of stress and pain on the development

of

416 the opioid system has also been studied. Footshock has no effect on binding but produces an analgesic ef-

[Met]-enkephalin levels in male pups at 70 and 120 days of age accompanied by a transient decrease in /3-

fect as evidenced

endorphin levels in the hypothalamus. The effect of perinatal low level lead exposure

by an increase in nociceptive

laten-

cy’. In addition Hamm and Knisely4’ have reported that analgesia induced by footshock is less in lo-dayold rats than in 28-day- and 5-7-month-old animals. Recurrent aqueductal

pain-related grey matter

stress, stimulation of perior repeated placement on a

hot plate, has been shown to increase opiate receptors and enkephalin levels in neonates”’ and chronic stress during

the first month

post-partum

has also

been found to increase the sensitivity of the adult to morphine analgesias”. In addition hot plate stress for l-2 h per day in preweanling rats causes a 25% reduction in naloxone binding in the midbrain, hypothalamus and striatum’i5. Thus it appears that the opioid system is involved with responses to stress and that stress during these important stages of development can affect the development of the system. It should be noted that opioid modulation of stress-induced rises in corticosterone is absent in rats in the early postnatal period. This effect matures extraordinarily late at about day 4.5 (ref. 7). 5.4.

Non-opioid

drugs

Drugs associated with other neurotransmitter systems administered during the perinatal period have also been found to affect the development of the opioid system. Dopamine receptor blockade with haloperidol during the prenatal period has been shown to decrease naloxone binding in the pups with a concomitant increase in dopamine bindings6 indicating a possible involvement of dopamine in opioid receptor development. 5,7-Dihydroxytryptamine, a serotonin neurotoxin, also decreases opioid binding’* as does prenatal diazepam exposure124. Capsaicin which degenerates unmyelinated axons, when given to the dam during pregnancy results in a decreased concentration of receptors in the spinal cord and a decreased affinity of receptors in the brainstem at birth7i. 5.5. Toxic chemicals The development of the opioid shown to be particularly sensitive to the insecticide, Chlordecone54 and lead6~74~75~K”~127. Chlordecone given tion of 1 mg/kg to 4-day-old rats,

system has been the toxic insult of the heavy metal, as a single injecreduces pituitary

has

been shown to alter various aspects of the opioid system. Both morphine75 and ketocyclazocine74 analgesia is decreased

in lo-day-old

tal proenkephalin products pressed by this exposure6.

rats and levels of striaare also markedly

de-

6. CONCLUSIONS/SUMMARY

The understanding of opioid system function in the adult has progressed markedly in the last decade. This in turn has led to an increased knowledge of how opioid function develops in the embryo and in the postnatal period. With respect to the ontogenetic profile of the opioid peptide products of the 3 opioid precursors, all are detectable during gestation and their development in rodents is not completed until well after birth, the third postnatal week often exhibiting the most marked increases. There is not an exact parallel development of the precursors and there may be regional fluctuations for some of the peptides in the postnatal period. All of the indications are that the ontogenesis of the 3 precursors occurs independently, and that their opioid peptide products have distinct ontogenetic profiles (Section 2.2.-2.4.). Indeed in a comparative immunocytochemical study for dynorphin-A and enkephalin36 the patterns of development in the hippocampal formation were dissimilar. In addition the differences observed in ontogenetic patterns for the precursor products may well reflect differential activities of the processing cleavage enzymes in the developing animal. Indeed a comparative ontogenetic study of ACTH and P-endorphin64%65showed a dissimilar profile during early development in rat brain and pituitary. Whilst these may be due to different processing patterns, the possibility of differential release, secretion or degradation of these peptides cannot be excluded and may be independent of the synthesis of the precursor. There is also a differential ontogeny of the opioid receptor subtypes and the recent evidence with highly selective binding ligands has provided a clear pattern. ,LL-And K-sites are the first to appear though 6receptors are absent until the second postnatal week

417 in the rat. In this species the full development

of p-

and b-sites occurs in the third and fourth weeks respectively. There is still some argument about the ontogenetic profile for x-sites though, like the &receptors, full development Ontogenetic

is probably later than for the p-sites. profiles of opioid receptors have also

been demonstrated in the chick, sheep and mouse. Too few studies have looked at peptide and receptor development in parallel and it is still unclear if specific peptide

development

is directly

linked with

development of a single receptor site. The postnatal development of both opioid

pep-

tides and receptors is mirrored by pharmacological actions of opioids which differ in the neonate. Their pharmacological effects are not solely dependent on the number of receptors but also on the ontogenetic pattern of the metabolising enzymes and on bloodbrain barrier development. There are some anomalous responses to opioids in the neonate and these in-

elude locomotion, respiratory depressant and effects on corticosterone release.

activity

There are also indications that the opioid system is particularly sensitive to insult during the developing period. In particular toxic effects at low doses have been shown for lead exposure, diazepam and haloperidol treatment. It may be that the complexity of opioid function increases the risk of disruption or it may reflect a ubiquitous involvement of the endogenous opioid peptides in the control of several neurochemicals in the brain. The future needs more studies in animals other than the rat and a clearer picture of the genetic profile of the precursors

and the processing

during development. long in coming.

This information

of the same should not be

ACKNOWLEDGEMENTS

J. McD . is supported

by The Wellcome

Trust.

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