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Effects of neonatal treatment with Tyr-MIF-1, morphiceptin, and morphine on development, tail flick, and blood-brain barrier transport
Det,elopmental Brain Research, 75 (19931 207-212 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00
2(17
BRESD 51692
Effects of neonatal treatment with Tyr-MIF-1, morphiceptin, and morphine on development, tail flick, and blood-brain barrier transport Laura M. Harrison, James E. Zadina, William A. Banks and Abba J. Kastin VA Medical Center and Tulane Unit,ersity School of Medicine and Neuroscience Program, New Orleans, I A 70146 (USA) (Accepted 18 May 1993)
Key words: Opiate; Antiopiate; Analgesia; Rat; Blood-brain barrier; Transport; Tyr-MIF-I; Morphine
Morphine and endogenous peptides can alter developmental processes, inducing changes that can endure into adulthood. Morphiceptin binds to mu opiate receptors and to non-opiate sites labeled by Tyr-MIF-I (Tyr-Pro-Leu-Gly-NH2), an endogenous brain peptide known to modulate opiate effects. Morphine, morphiceptin, Tyr-MIF-1, morphine + Tyr-MIF-1, and morphiceptin + Tyr-MIF-1 (5(1/xg, s.c.) were given to rats during their first week of life. Animals given morphine alone or in combination with Tyr-MIF-I had significantly lower body weights for the first 3 weeks of life and delayed eye opening on day 16. Rats given morphine had hypersensitive tail flick responses on day 9 while those given morphine +Tyr-MIF-1 were hypersensitive on days 3, 8, and 9. Locomotor, passive avoidance, and rotorod behaviors were not altered by the neonatal treatments. Transport of [1251]Tyr-MIF-1 out of the brain was tested on day 23 and found to be increased by neonatal morphine, an effect that was significantly potentiated by neonatal Tyr-MIF-I. The results indicate that neonatal administration of pcptides and opiates can affect later peptide transport across the blood brain barrier as well as selected developmental characteristics.
INTRODUCTION
The development of the nervous system can be altered by the administration of some peptides during the perinatal period. Behavioral and physiological changes, such as increased motor activity, decreased emotionality, and increased weights of ovaries, testes, pineal gland, and hypothalamus, result from neonatal treatment with thyrotropin releasing hormone 2t. These effects persist long after the end of the treatment period. Neonatal c~-MSH or its 4-10 amino acid core alters social behavior, performance on learning tasks 6'7 and muscle maturation Lls'l~. Administration of /3-endorphin during the perinatal period alters mu opiate receptors on day 14 24 and during the neonatal period increases sensitivity to thermal pain 23'27, a change that can endure into adulthood 27. This hypersensitivity is paradoxical to the immediate antinociceptive effect usually induced by opiates. Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 21 and MIF-1 (ProLeu-Gly-NH 2), peptides with opiate-modulating properties 11,12.15.25, also have been used in perinatal studies.
MIF-1 given postnatally s and Tyr-MIF-I given prenatally 24 resulted in higher body weights, an effect opposite to that produced by opiates. Tyr-MIF-1 also was able to block the hypersensitivity to thermal pain induced by /3-endorphin when the two peptides were co-administered during the first week of life 27. In the study reported here, we tested whether Tyr-MIF-1 could modify the effects of two other opiate agonists, morphine and morphiceptin, on several developmental measures. Tyr-MIF-1 and Met-enkephalin are transported across the blood-brain barrier (BBB) in the direction of CNS to blood by the same saturable carrier-mediated transport system, termed peptide transport system-1 (PTS-1) 2'4. This system is stereospecific, does not transport peptides in the blood to brain direction, and is altered in senescence. /3-Endorphin and morphiceptin are not transported by this system 4. Changes in the transport rate of PTS-1 have been associated with changes in nociception:. Although many studies have examined the development of the endothelial cells comprising the BBB, the development of transport
Correspondence: L.M. Harrison, VA Medical Center, Research Service (151), 1601 Perdido St, New Orleans, LA 7(1146, USA. Fax: (1) (504) 522-8559.
208 systems across the BBB has been less well studied. The BBB of rodents
is i n t a c t b y t h e t i m e o f b i r t h , b u t t h e
perinatal
period
saturable
transport
known and
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is o n e several
are transported
treatment
with
of dynamic
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developmental during
the
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opiates
the are
characteristics
by PTS-1, we examined
opiates
would affect the development
MATERIALS
changes
Because
neonatal
whether period
of PTS-1.
AND METHODS
Holtzman female rats arrived at our laboratory on day 8 of pregnancy and were housed singly under a 12:12 light:dark cycle, with lights on from 06.00 to 18.00 h. On the day of birth (day 1), male and female pups were assigned to receive 10 ~1 of the diluent control vehicle (0.9% NaCI, 0.01 M acetic acid) or one of the following coded solutions (50 t t g / p u p / c o m p o u n d ) : morphiceptin, morphine sulfate, Tyr-MIF-1, morphiceptin +Tyr-MIF-1, morphine sulfate + TYr-MIF-I. In order to randomize assignment to treatment groups, pups were ranked by weight within their litters, and each litter was randomly assigned to a sequence of injection. Each pup had littermate controls, and each treatment group appeared equally as often as each other treatment group at a given weight. Injections were made daily for the first 7 days of life with a 25-gauge, 2.5 cm needle. To prevent leakage of the injectate out of the pup after the s.c. injection, the needle was inserted at the base of the tail, and the injectate was deposited at the nape of the neck. The first injection was given between 6.00 and 8.75 h after birth, and the second injection was given 13 to 21 h later. All pups were weighed daily and were inspected for eye opening beginning on day 13. All measurements were made by an observer who did not know which solution had been administered.
40% urethane (2 g / k g injected i.p. m a volume o! 10 ml/kg), and the skulls were exposed. A hole was made in the skull with ~t guarded 26-gauge needle at a position 1 mm lateral and 1 mm posterior to the bregma. A Hamilton syringe was used to inject into the right lateral ventricle 1.0 /xl of lactated ringer's solution containing 25,000 CPM of [t25I]Tyr-MlF-l. Rats were decapitated at 2 ~i 10 rain aftcl injection. Brains were removed, weighed, and counted in a gamma counter for 3 min. Transport rate at 10 rain was determined by the equation T=(A-M)/I.
where T is the CPM transported per minute by whole brain, M the number of counts remaining in the individual brains, t the time in minutes from injection to decapitation, and A the amount of material that is available for transport. The value A is the antilog of the intercept of the relationship between log M (as the y-value) and t. Statistical measures
Group differences were tested with analysis of variance (ANOVA) followed, where appropriate, by Duncan's Multiple Range Test. When no sex differences were detected after initial analysis, data were collapsed over sex for further analyses. RESULTS
Body weight Fig. 1 shows results of measurement Although
the groups
were
matched
of body weight. for weight
at the
60 Behavioral measures Tail flick The distal half of the tail was placed in warm water baths of increasing temperature (37, 38, 39, 40, 41, 42, 43, and 44°C) for 5 s or until the pup removed it. The dependent measure was the temperature at which the tail was first removed before the maximal time had elapsed. Two successive trials were done each day for 5 non-consecutive days (days 3, 4, 5, 8, and 9) and the mean of the two trials was used for analysis. On days when the pups were still receiving injections, the injections were given immediately after the two tail flick trials.
50 ¸
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~
30-
o'~ 20m Other behavioral measures Groups of pups were tested on several behavioral measures during the second 2 weeks of life. Activity was determined from the number of lines crossed in a 70 mm X 70 mm grid and the amount of time spent in the center or the periphery. Rotorod 27 and passive avoidance I3 tests were conducted as previously described except that shock was omitted in the rotorod test. Briefly, in the rotorod test, rat pups were tested for motor coordination using a Columbus Instruments Rotomex rotorod. The latency to fall from a 7.5 cm diameter bar rotating at a speed of 10 rpm was recorded in a single trial on each of two consecutive days. In the passive avoidance test, rat pups were placed on an 11 mm x 7.5 mm x 2.5 mm (lenth × width × height) box, and the latency to step down was recorded. After receiving a brief, mild shock, the animal was returned to the box, and the latency to step down was recorded in three consecutive trials. Brain-to-blood transport o f [1251]Tyr-M1F-1 on day 23
The method for determination of brain-to-blood transport was previously described 3. Briefly, 23-day-old rats were anesthetized with
10-
0
o
1'o
1'5
ao
a5
Day Fig. 1. Body weight for days 1, 3, 5, 7, 14, 21. On days 3, 5, 7, 14, and 21, morphine+Tyr-MIF-1 caused significantly lower body weights than control pups ( P < 0.01). The body weights for morphine + TyrMIF-1 treated pups on those days were (mean _+S.D.): 8.75_+0.85, 10.86_+1.47, 13.34_+1.82, 28.72_+3.61, 48.10+6.00, and those for control animals were 9.62+ 1.03, 12.49-+ 1.54, 15.89+ 1.99, 32.47-+ 3.14, 53.16-+6.20. On days 3, 5, 7, and 14, pups treated with morphine had significantly lower body weights ( P < 0.05). Body weights for morphine-treated animals were (mean-+ S.D.): 9.02 + 0.82, 1 t.29 _+ 1.21, 14.00-+ 1.59, 30.20-+2.66. Dil, diluent; TM, Tyr-MIF-1; MCP, morphiceptin; M C P + TM, morphiceptin +Tyr-MIF-li MS, morphine sulfate; MS + TM, morphine sulfate + Tyr-MIF- t.
209
beginning of the injections, the effect of treatment was highly significant in the overall repeated measures analysis (F5,12 2 = 7.37; P < 0.0001) as well as in the individual analyses on each day: F5,12 2 = 5.73, P < 0.001 for day 3; F5,12 2 = 6.34, P < 0.0001 for day 5; F,;,12 2 = 8.73, P < 0.0001 for day 7; F5,12 2 = 7 . 0 , P < 0.0001 for day 14 and Fs,122 = 4.33, P < 0.01 for day 21. In comparison tests subsequent to the ANOVAs, the combination of morphine + Tyr-MIF-1 was shown to significantly lower body weight on each day tested ( P < 0.01 ). Morphine alone caused significantly lower body weights on days 3, 5, 7, and 14, but not on day 21. The weights of animals given morphine + Tyr-MIF-1 were significantly lower than those of controls on day 21 ( P < 0.01 ), when the difference between weights of control and morphine-treated pups was no longer significant.
Eye opening Eye opening was measured as the proportion of pups with eyes open on a given day. None of the pups had open eyes on day 13, the day on which inspection began. By day 16, a significant (Fs,,~5 = 3.61; P < 0.01) effect of treatment was evident as shown in Fig. 2. There was a significantly lower proportion of pups with eyes open in the morphine ( P < 0.01) and morphine + Tyr-MIF-1 ( P < 0.05) groups than in the diluent control group.
Tail flick Morphine alone and the combination of morphine + Tyr-MIF-1 caused pups to remove their tails at a lower bath t e m p e r a t u r e than control animals, Fig. 3
TAIL FLICK ON DAY 9 42.442.2~: 42.0 41.8 (.9 z 41.6 <
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5
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40.8
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1 D'IL
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Fig. 3. Tail flick responses on day 9. Mean ( + S.E.) bath tempera tures are shown. Pups pretreated with m o r p h i n c ( * :+: 17- 0.01) or m o r p h i n e + T y r - M l F - I (*P
shows the values for day 9, the last day tested. Both thc morphine-treated pups ( P < 0.01 ) and the morphine + T y r - M I F - l - t r e a t e d pups ( P < 0.05) removcd their tails at significantly lower bath temperatures than control animals. Morphine alone also tended to cause tail removal at a lower temperature than controls on day 3 ( P = 0.059). On the earlier days, the only othcr group with significant differences from thc control was that receiving the combination of morphinc + T y r - M I F - I : this treatment caused pups to remove their tails at at significantly ( P < 0.05) lower bath tcmperature on days 3 and 8, in addition to day 9.
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Fig. 2. Eye opening on day ]6, Results are expressed as proportion of pups in each group (+_S.E.) with eyes open on this day. The groups receiving morphine+Tyr-MIF-1 (* P < 0.05) or morphine ( * * P < 0.0l) had fewer pups with eyes open on this day than the control group.
~.
600-
ca. 0
4002000
DIL
TM
MCP MCP+TM MS MS+TM group Fig. 4. Transport of [taSl]Tyr-MIF-I out ,,ff the brain ,.m day 23. Results arc given as CPM transported o u t / b r a i n / m i n . Pups gp,'cn morphine +Tyr-MIF-1 ( * * P <" 0.01)or morphine ahme ( +: P < 0.{151 had faster transport than controls.
210 Other behavioral tests There were no significant group differences in the tests of activity, passive avoidance, or rotorod behavior. Brain-to-blood transport of [1251]Tyr-MIF-1 on day 23 Fig. 4 shows that neonatal treatment with morphine alone significantly (P < 0.05) enhanced brain-to-blood transport of [~25I]Tyr-MIF-1 on the day of testing (day 23). This represented an 89% enhancement of transport rate. Pretreatment with morphine + Tyr-MIF-1 caused a 200% increase in transport rate over that of control animals (P < 0.001). Transport in the morphine + Tyr-MIF-1 group was significantly faster than in pups given morphine alone ( P < 0.05) or Tyr-MIF-1 alone (P < 0.01). DISCUSSION The main findings of this study are: (a) neonatal morphine decreased body weight, and this effect was enhanced when morphine was given in combination with Tyr-MIF-1; (b) both morphine and morphine + Tyr-MIF-1 caused a delay in eye opening; (c) morphine and morphine + Tyr-MIF-1 caused hypersensitivity in the tail flick test; and (d) pretreatment with morphine increased transport of [125I]Tyr-M1F-1 out of the brain on day 23, an effect that was significantly greater in rats treated with the combination of morphine + TyrMIF-1. Thus, Tyr-MIF-1 can enhance morphine-induced changes in neonatally treated rat pups, and these effects persist after the treatment period. The results are consistent with the recent observations in the guinea pig ileum assay that Tyr-MIF-1 can act as an opiate agonist under certain conditions 25 in addition to its originally defined role as an opiate antagonist 12'25. The antagonist effects of Tyr-MIF-1 have been best observed when the peptide was given after chronic rather than acute treatment with morphine 25. The current study indicates that when Tyr-MIF-1 is co-administered with morphine for several days, it does not antagonize the effects of the chronic morphine, at least at the ages and doses tested here. The finding that morphine lowered body weight was expected. Previous studies 9'1°'16'2°'2s have shown that morphine can inhibit somatic development. In addition, studies using the opiate antagonist naltrexone have implicated endogenous opiates in the regulation of growth. Doses of naltrexone greater than 10 mg/kg daily from birth to day 21, which block the opiate receptor for 24 h/day, enhance somatic development, while lower doses inhibit development 29-31. Tyr-MIF-1 binds to its own non-opiate receptor and at higher doses, to the opiate receptor 22'26 where its
actions are complex: it can act as an antagonist when the receptor reserve is relatively low, but as an agonist in conditions of high receptor reserve-". Tyr-MIF-I alone, when administered prenatally, caused a significant increase in pup body weight on day 7. an antiopiate-like effect 24. In this study and in previous ones 23'24'27, Tyr-MIF-1 given in the postnatal period did not affect body weight. It is possible that a loss of the antiopiate effect of Tyr-MIF-I in the growing animal reflects the development of receptors and their coupling processes, resulting in a greater agonist-like activity of the peptide. Alternatively, these findings may be related to the Tyr-MIF-1 induced changes in the plasma concentration of growth hormone reported in adult rats 17. In contrast to the effects of morphine, two peptide agonists,/3-endorphin and morphiceptin, did not affect body weight when administered alone or in combination with Tyr-M1F-1 during the first week of life 27. In the present study, Tyr-MIF-1 not only failed to antagonize the effect of morphine, but slightly potentiated it. These effects of morphine and morphine + Tyr-MIF-I lasted at least 2 weeks beyond the neonatal period of treatment. Thus, although Tyr-MIF-1 can stimulate somatic development if given during the prenatal period 24, this antiopiate-like effect is no longer present if it is given postnatally when opiate receptors are more fully developed. As with body weight, Tyr-MIF-I neither affected eye opening by itself nor antagonized the delayed eye opening caused by morphine. The opiate antagonist naltrexone has been shown to accelerate eye opening when given at a higher dose (50 mg/kg) and for a longer time (days 0-21) than the test substances given in our study -~°. Similarly, MIF-1, an antiopiate peptide apparently without the opiate effects of Tyr-MIF-1, caused early eye opening in mice when given at a dose of 1 mg/kg on days 2-19 s. Thus, it is possible that Tyr-MIF-1 might have exerted an antiopiate effect if given for a longer time, but it is also possible that its opiate agonist actions would preclude such a change. Our group of pups receiving morphine on days 1-7 had a smaller proportion with eyes open on day 16 than did the control pups. In other studies 23'27, /3-endorphin, injected at doses and over the time identical to that of the present study, had no effect on eye opening. Pups pretreated with morphine were hypersensitive in the tail flick test on day 9. Tyr-MIF-1 did not antagonize this effect, nor did it affect the tail flick response when administered alone. In a previous study, neonatally administered /3-endorphin was shown to cause hypersensitivity in the tail flick test on day 9. However, this effect was antagonized by Tyr-MIF-127.
211
The present study, therefore, highlights the possible differences in the mechanisms of morphine-induced and /3-endorphin-induced hypersensitivity in the tail flick test and their differing susceptibilities to antagonism by the opiate-modulating peptide Tyr-M1F-1. The effects of morphine and Tyr-MIF-1 on the BBB transport system PTS-1 further illustrate the peptide's opiate-modulating properties. Although neonatally injected Tyr-MIF-1 did not have a significant effect by itself, it significantly potentiated morphine's effect of enhancing transporf of the labeled peptide out of the brain. Morphine previously had been shown to inhibit this rate of transport in adult mice when administered either acutely or chronically 5. However, that study measured transport 1 h after administration of morphine, whereas the present study measured it 2 weeks after administration of the drug had ended. Furthermore, paradoxical effects often occur with neonatal, as opposed to adult, administration of drugs. A long-term upregulation of transporters, induced by neonatal treatment, is implied. The presence of exogenous peptide or drug during a critical period could cause the development of an enhanced mechanism for removal, probably involving carrier-mediated transport through the BBB. The modulating site appears to be susceptible to both morphine and Tyr-M1F-1. In summary, neonatal administration of morphine can change, in a manner that Tyr-MIF-1 can in some cases potentiate, several developmental processes including somatic development, nociception, eye opening, and peptide transport across the blood-brain barrier.
Acknowled~q,ernents. This work was supported by the VA.
REFERENCES 1 Acker, G.R., Berran, J. and Strand, F.L. (1985) A C T H neuromodulation of the developing motor system and neonatal learning in the rat, Peptides, 6, 41 49. 2 Banks, W.A. and Kastin, A.J. (1987) Minireview: saturable transport of peptides across the blood-brain barrier, Lift" Sci., 41, 1319-1338. 3 Banks, W.A. and Kastin, A.J. 11989) Quantifying carrier-mediated transport of peptides from the brain to the blood. In P.M. Conn (Ed.), Methods in Enzyrnology, Vol. 168, Academic Press, San Diego, pp. 652-660. 4 Banks, W.A. and Kastin, A.J. 11990) Editorial review: peptidc transport systems for opiates across the blood brain barrier, Am. J. Physiol., 259, E l - E l ( ) . 5 Banks, W.A., Kastin, A.J. and Nager, B.J. 11988) Analgesia and the blood-brain barrier transport system for T y r - M I F - 1 / e n kephalins: evidence for a dissociation, Neuropharmaeology, 27, 175 179. 6 Beckwith, B.E., O'Quin, R.K., Petro, M.S., Kastin, A.J. and Sandman, C.A. (1977) The effect of neonatal injections of alpha-
MSH on the open field behavior of juvenile and adult rats, Physiol. Psychol., 5, 295-299. 7 Beckwith, B.E., Sandman, C.A., Hothersall, D. and Kaslin, A.J. 11977) Influence of neonatal injections of alpha-MSH on learning, memory,, and attention in rats, Physiol Behar., 18, 63 71. 8 D'Amore, A., Pieretti, S., Palazzesi, S., Pezzmi, G., Chiaroni, F., Scorza, T. and Loizzo, A. (199(I) MIF-I can accelerate neuromolot, E E G and behavioral development in mice, Peptides, 11, 527 532. 9 Davis, W.M. and Lin, C.H. (1972) l'renatal morphine effects on survival and behavior of rat offspring, Rcs. ('ommun. (Ttem. Pathol. Pharmacol., 3, 2115-214. 111 Friedler, G. and Cochin, J. 11972) Growth retardation in offspring of female rats treated with morphine prior to conception, Science. 175. 654 656. 11 Galina, Z.H. and Kaslin, A.J. 11986) Existence of antiopiate systems as illustrated by M I F - I / T y r - M 1 F - I , Li[~, Sci., 39, 21532159. 12 Galina, Z.H. and Kastin, A.J. (1987)Tyr-MIF-I attenuates antinociceptive responses induced by three models of stress-analgesia, Br. J. Pharmacol., 90, 669-674. 13 Hayashi, T., Kastin, A.J., Coy, D.It. and Olson, R.I). 11983) Tyr-MIF-I affects passive aw~idance behavior but nnt motor activity in rats, Brain Res. Bull., I I, 659 662. 14 Johanson, C.E. (1989) Ontogeny and phylogeny of the bloodbrain barrier. In G.A. Neuwelt (Ed.), Implications o( the' Illood-. brain Barrier and its" Manipulation, Vol. 1., Ba,~ic Science Aspects, Plenum, New York, pp. 157 198. 15 Kaslin, A.J., Olson, R.D., Ehrensing, R.H., Berzas, M.('., Schally, A.V. and Coy, D,H. (1979) MIF-I's differential actions as an opiate antagonist. Pharmacol. Biochem. Behar., I I, 721 723. 16 Kirby, M . L (1983) Recovery of spinal cord volume in postnatal rats fl)llowing prenatal exposure to morphine, l)et'. Brain Res., 6, 211 217. 17 Krowicki. Z.K. and Kastin, A.J. 11991) Effects of Tyr-MIF-I (Tyr-Pro-Leu-Gly-NHe) on GH, LH, prolactin, and TSH secretion with and without morphine, NeuropeptMes, 18, 41-47. 18 Rose, K.J., Frischer, R.E., King, J.A. and Strand, F.L. 11988) Neonatal neuromuscular parameters vaD' in susceptibility to postnatal A C T H / M S t i 4 l0 administration, Peptides, 9. 151 156. 19 Rose, K.J. and Strand, F.L. (199(t) A ( ' T H / M S t i ( 4 Ill) peptidc increases postnatal metabolic activity of EDL muscle following early prenatal administration, t'{7~tides. 1 I, 177-179. 211 Sobrian, S.K. (1977) Prenatal morphine administration alters behavioral development in the rat, Pharmacol. Biochem. Behat',, 7, 285-288. 21 Stratton, L.O., Gibson, C.A., Kolar, K.G. and Kastin, A.J. (1976) Neonatal treatment with T R t I affects development, learning, and emotionality in the rat, Pharmacol. Biochem. Behat., Suppl. I, 5, 65 67. 22 Zadina, J.E. and Kastin, A.J. (1985) Interactions between the antiopiate Tyr-MIF-1 and the mu opiate morphiccptin at their respective binding sites in brain, PeptMes, 6, 965-970. 23 Zadina. J.E. and Kastin, A.J. (1986} Nconalal peptidcs affect developing rats: /3-endorphin alters nociception and opiate receptors, corticotrophm-releasing factor alters corticosteronc, Det. Brain Res., 29, 21-29. 24 Zadina. J.E., Kastin, A.J., ('oy, D.}I. and Adinoff, B.A. (1985) Developmental, behavioral, and opiate receptor changes after prenatal or pnstmttal /3-endorphin, CRF, or Tyr-MIF-I, f{~'ychoneuroendoerinology, 10, 367-383. 25 Zadina, J.E., Kastin, A.J., Kersh, D. and Wyatt, A. (1992) TyrMIF-1 and hemorphin can act :is opiate agonists as well as antagonists in the guinea pig ileum, Lil? Sci., 51,869 885. 26 Zadina, J.E., Kastin, A.J., Krieg, E.F. and Coy, D,tt. (1982) Characterization of binding sites for N-Tyr-M1F-I (Tyr-Pro-LeuGIy-NH ) in rat brain, Phannaeol. Biochem. Behar,, 17, 1193 1 ITS8. 27 Zadina, J.E., Kastin, A.J.. Manasco, P.K., Pignaticllo, M.F, and Nastiuk, K.L. (1987) Long-term hyperalgesia induced by neonatal ¢3-cndorphin and morphiceptin is blocked by neonatal Tyr-MIF-1, Brain Res., 409, 10-18.
212 28 Zagon, I.S. and McLaughlin, P.J. (1977) Morphine and brain growth retardation in the rat, Pharmacology, 15, 276-282. 29 Zagon, I.S. and McLaughlin, P.J. (1983) Increased brain size and cellular content in infant rats treated with an opiate antagonist, Science, 221, 1179-1180.
30 Zagon, I.S. and McLaughlin, P,J. (1983) Naltrcxone modulates growth in infant rats, Life Sci., 33, 2449-2454. 31 Zagon, I.S. and McLaughlin, P.J. (1984) Naltrexone modulates body and brain development in infant rats: a role for endogenous c~pioid systems in growth, L!fe Sci,, 35, 2057-21)~4.
2(17
BRESD 51692
Effects of neonatal treatment with Tyr-MIF-1, morphiceptin, and morphine on development, tail flick, and blood-brain barrier transport Laura M. Harrison, James E. Zadina, William A. Banks and Abba J. Kastin VA Medical Center and Tulane Unit,ersity School of Medicine and Neuroscience Program, New Orleans, I A 70146 (USA) (Accepted 18 May 1993)
Key words: Opiate; Antiopiate; Analgesia; Rat; Blood-brain barrier; Transport; Tyr-MIF-I; Morphine
Morphine and endogenous peptides can alter developmental processes, inducing changes that can endure into adulthood. Morphiceptin binds to mu opiate receptors and to non-opiate sites labeled by Tyr-MIF-I (Tyr-Pro-Leu-Gly-NH2), an endogenous brain peptide known to modulate opiate effects. Morphine, morphiceptin, Tyr-MIF-1, morphine + Tyr-MIF-1, and morphiceptin + Tyr-MIF-1 (5(1/xg, s.c.) were given to rats during their first week of life. Animals given morphine alone or in combination with Tyr-MIF-I had significantly lower body weights for the first 3 weeks of life and delayed eye opening on day 16. Rats given morphine had hypersensitive tail flick responses on day 9 while those given morphine +Tyr-MIF-1 were hypersensitive on days 3, 8, and 9. Locomotor, passive avoidance, and rotorod behaviors were not altered by the neonatal treatments. Transport of [1251]Tyr-MIF-1 out of the brain was tested on day 23 and found to be increased by neonatal morphine, an effect that was significantly potentiated by neonatal Tyr-MIF-I. The results indicate that neonatal administration of pcptides and opiates can affect later peptide transport across the blood brain barrier as well as selected developmental characteristics.
INTRODUCTION
The development of the nervous system can be altered by the administration of some peptides during the perinatal period. Behavioral and physiological changes, such as increased motor activity, decreased emotionality, and increased weights of ovaries, testes, pineal gland, and hypothalamus, result from neonatal treatment with thyrotropin releasing hormone 2t. These effects persist long after the end of the treatment period. Neonatal c~-MSH or its 4-10 amino acid core alters social behavior, performance on learning tasks 6'7 and muscle maturation Lls'l~. Administration of /3-endorphin during the perinatal period alters mu opiate receptors on day 14 24 and during the neonatal period increases sensitivity to thermal pain 23'27, a change that can endure into adulthood 27. This hypersensitivity is paradoxical to the immediate antinociceptive effect usually induced by opiates. Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 21 and MIF-1 (ProLeu-Gly-NH 2), peptides with opiate-modulating properties 11,12.15.25, also have been used in perinatal studies.
MIF-1 given postnatally s and Tyr-MIF-I given prenatally 24 resulted in higher body weights, an effect opposite to that produced by opiates. Tyr-MIF-1 also was able to block the hypersensitivity to thermal pain induced by /3-endorphin when the two peptides were co-administered during the first week of life 27. In the study reported here, we tested whether Tyr-MIF-1 could modify the effects of two other opiate agonists, morphine and morphiceptin, on several developmental measures. Tyr-MIF-1 and Met-enkephalin are transported across the blood-brain barrier (BBB) in the direction of CNS to blood by the same saturable carrier-mediated transport system, termed peptide transport system-1 (PTS-1) 2'4. This system is stereospecific, does not transport peptides in the blood to brain direction, and is altered in senescence. /3-Endorphin and morphiceptin are not transported by this system 4. Changes in the transport rate of PTS-1 have been associated with changes in nociception:. Although many studies have examined the development of the endothelial cells comprising the BBB, the development of transport
Correspondence: L.M. Harrison, VA Medical Center, Research Service (151), 1601 Perdido St, New Orleans, LA 7(1146, USA. Fax: (1) (504) 522-8559.
208 systems across the BBB has been less well studied. The BBB of rodents
is i n t a c t b y t h e t i m e o f b i r t h , b u t t h e
perinatal
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opiates
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MATERIALS
changes
Because
neonatal
whether period
of PTS-1.
AND METHODS
Holtzman female rats arrived at our laboratory on day 8 of pregnancy and were housed singly under a 12:12 light:dark cycle, with lights on from 06.00 to 18.00 h. On the day of birth (day 1), male and female pups were assigned to receive 10 ~1 of the diluent control vehicle (0.9% NaCI, 0.01 M acetic acid) or one of the following coded solutions (50 t t g / p u p / c o m p o u n d ) : morphiceptin, morphine sulfate, Tyr-MIF-1, morphiceptin +Tyr-MIF-1, morphine sulfate + TYr-MIF-I. In order to randomize assignment to treatment groups, pups were ranked by weight within their litters, and each litter was randomly assigned to a sequence of injection. Each pup had littermate controls, and each treatment group appeared equally as often as each other treatment group at a given weight. Injections were made daily for the first 7 days of life with a 25-gauge, 2.5 cm needle. To prevent leakage of the injectate out of the pup after the s.c. injection, the needle was inserted at the base of the tail, and the injectate was deposited at the nape of the neck. The first injection was given between 6.00 and 8.75 h after birth, and the second injection was given 13 to 21 h later. All pups were weighed daily and were inspected for eye opening beginning on day 13. All measurements were made by an observer who did not know which solution had been administered.
40% urethane (2 g / k g injected i.p. m a volume o! 10 ml/kg), and the skulls were exposed. A hole was made in the skull with ~t guarded 26-gauge needle at a position 1 mm lateral and 1 mm posterior to the bregma. A Hamilton syringe was used to inject into the right lateral ventricle 1.0 /xl of lactated ringer's solution containing 25,000 CPM of [t25I]Tyr-MlF-l. Rats were decapitated at 2 ~i 10 rain aftcl injection. Brains were removed, weighed, and counted in a gamma counter for 3 min. Transport rate at 10 rain was determined by the equation T=(A-M)/I.
where T is the CPM transported per minute by whole brain, M the number of counts remaining in the individual brains, t the time in minutes from injection to decapitation, and A the amount of material that is available for transport. The value A is the antilog of the intercept of the relationship between log M (as the y-value) and t. Statistical measures
Group differences were tested with analysis of variance (ANOVA) followed, where appropriate, by Duncan's Multiple Range Test. When no sex differences were detected after initial analysis, data were collapsed over sex for further analyses. RESULTS
Body weight Fig. 1 shows results of measurement Although
the groups
were
matched
of body weight. for weight
at the
60 Behavioral measures Tail flick The distal half of the tail was placed in warm water baths of increasing temperature (37, 38, 39, 40, 41, 42, 43, and 44°C) for 5 s or until the pup removed it. The dependent measure was the temperature at which the tail was first removed before the maximal time had elapsed. Two successive trials were done each day for 5 non-consecutive days (days 3, 4, 5, 8, and 9) and the mean of the two trials was used for analysis. On days when the pups were still receiving injections, the injections were given immediately after the two tail flick trials.
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o'~ 20m Other behavioral measures Groups of pups were tested on several behavioral measures during the second 2 weeks of life. Activity was determined from the number of lines crossed in a 70 mm X 70 mm grid and the amount of time spent in the center or the periphery. Rotorod 27 and passive avoidance I3 tests were conducted as previously described except that shock was omitted in the rotorod test. Briefly, in the rotorod test, rat pups were tested for motor coordination using a Columbus Instruments Rotomex rotorod. The latency to fall from a 7.5 cm diameter bar rotating at a speed of 10 rpm was recorded in a single trial on each of two consecutive days. In the passive avoidance test, rat pups were placed on an 11 mm x 7.5 mm x 2.5 mm (lenth × width × height) box, and the latency to step down was recorded. After receiving a brief, mild shock, the animal was returned to the box, and the latency to step down was recorded in three consecutive trials. Brain-to-blood transport o f [1251]Tyr-M1F-1 on day 23
The method for determination of brain-to-blood transport was previously described 3. Briefly, 23-day-old rats were anesthetized with
10-
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1'o
1'5
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Day Fig. 1. Body weight for days 1, 3, 5, 7, 14, 21. On days 3, 5, 7, 14, and 21, morphine+Tyr-MIF-1 caused significantly lower body weights than control pups ( P < 0.01). The body weights for morphine + TyrMIF-1 treated pups on those days were (mean _+S.D.): 8.75_+0.85, 10.86_+1.47, 13.34_+1.82, 28.72_+3.61, 48.10+6.00, and those for control animals were 9.62+ 1.03, 12.49-+ 1.54, 15.89+ 1.99, 32.47-+ 3.14, 53.16-+6.20. On days 3, 5, 7, and 14, pups treated with morphine had significantly lower body weights ( P < 0.05). Body weights for morphine-treated animals were (mean-+ S.D.): 9.02 + 0.82, 1 t.29 _+ 1.21, 14.00-+ 1.59, 30.20-+2.66. Dil, diluent; TM, Tyr-MIF-1; MCP, morphiceptin; M C P + TM, morphiceptin +Tyr-MIF-li MS, morphine sulfate; MS + TM, morphine sulfate + Tyr-MIF- t.
209
beginning of the injections, the effect of treatment was highly significant in the overall repeated measures analysis (F5,12 2 = 7.37; P < 0.0001) as well as in the individual analyses on each day: F5,12 2 = 5.73, P < 0.001 for day 3; F5,12 2 = 6.34, P < 0.0001 for day 5; F,;,12 2 = 8.73, P < 0.0001 for day 7; F5,12 2 = 7 . 0 , P < 0.0001 for day 14 and Fs,122 = 4.33, P < 0.01 for day 21. In comparison tests subsequent to the ANOVAs, the combination of morphine + Tyr-MIF-1 was shown to significantly lower body weight on each day tested ( P < 0.01 ). Morphine alone caused significantly lower body weights on days 3, 5, 7, and 14, but not on day 21. The weights of animals given morphine + Tyr-MIF-1 were significantly lower than those of controls on day 21 ( P < 0.01 ), when the difference between weights of control and morphine-treated pups was no longer significant.
Eye opening Eye opening was measured as the proportion of pups with eyes open on a given day. None of the pups had open eyes on day 13, the day on which inspection began. By day 16, a significant (Fs,,~5 = 3.61; P < 0.01) effect of treatment was evident as shown in Fig. 2. There was a significantly lower proportion of pups with eyes open in the morphine ( P < 0.01) and morphine + Tyr-MIF-1 ( P < 0.05) groups than in the diluent control group.
Tail flick Morphine alone and the combination of morphine + Tyr-MIF-1 caused pups to remove their tails at a lower bath t e m p e r a t u r e than control animals, Fig. 3
TAIL FLICK ON DAY 9 42.442.2~: 42.0 41.8 (.9 z 41.6 <
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Fig. 3. Tail flick responses on day 9. Mean ( + S.E.) bath tempera tures are shown. Pups pretreated with m o r p h i n c ( * :+: 17- 0.01) or m o r p h i n e + T y r - M l F - I (*P
shows the values for day 9, the last day tested. Both thc morphine-treated pups ( P < 0.01 ) and the morphine + T y r - M I F - l - t r e a t e d pups ( P < 0.05) removcd their tails at significantly lower bath temperatures than control animals. Morphine alone also tended to cause tail removal at a lower temperature than controls on day 3 ( P = 0.059). On the earlier days, the only othcr group with significant differences from thc control was that receiving the combination of morphinc + T y r - M I F - I : this treatment caused pups to remove their tails at at significantly ( P < 0.05) lower bath tcmperature on days 3 and 8, in addition to day 9.
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Fig. 2. Eye opening on day ]6, Results are expressed as proportion of pups in each group (+_S.E.) with eyes open on this day. The groups receiving morphine+Tyr-MIF-1 (* P < 0.05) or morphine ( * * P < 0.0l) had fewer pups with eyes open on this day than the control group.
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MCP MCP+TM MS MS+TM group Fig. 4. Transport of [taSl]Tyr-MIF-I out ,,ff the brain ,.m day 23. Results arc given as CPM transported o u t / b r a i n / m i n . Pups gp,'cn morphine +Tyr-MIF-1 ( * * P <" 0.01)or morphine ahme ( +: P < 0.{151 had faster transport than controls.
210 Other behavioral tests There were no significant group differences in the tests of activity, passive avoidance, or rotorod behavior. Brain-to-blood transport of [1251]Tyr-MIF-1 on day 23 Fig. 4 shows that neonatal treatment with morphine alone significantly (P < 0.05) enhanced brain-to-blood transport of [~25I]Tyr-MIF-1 on the day of testing (day 23). This represented an 89% enhancement of transport rate. Pretreatment with morphine + Tyr-MIF-1 caused a 200% increase in transport rate over that of control animals (P < 0.001). Transport in the morphine + Tyr-MIF-1 group was significantly faster than in pups given morphine alone ( P < 0.05) or Tyr-MIF-1 alone (P < 0.01). DISCUSSION The main findings of this study are: (a) neonatal morphine decreased body weight, and this effect was enhanced when morphine was given in combination with Tyr-MIF-1; (b) both morphine and morphine + Tyr-MIF-1 caused a delay in eye opening; (c) morphine and morphine + Tyr-MIF-1 caused hypersensitivity in the tail flick test; and (d) pretreatment with morphine increased transport of [125I]Tyr-M1F-1 out of the brain on day 23, an effect that was significantly greater in rats treated with the combination of morphine + TyrMIF-1. Thus, Tyr-MIF-1 can enhance morphine-induced changes in neonatally treated rat pups, and these effects persist after the treatment period. The results are consistent with the recent observations in the guinea pig ileum assay that Tyr-MIF-1 can act as an opiate agonist under certain conditions 25 in addition to its originally defined role as an opiate antagonist 12'25. The antagonist effects of Tyr-MIF-1 have been best observed when the peptide was given after chronic rather than acute treatment with morphine 25. The current study indicates that when Tyr-MIF-1 is co-administered with morphine for several days, it does not antagonize the effects of the chronic morphine, at least at the ages and doses tested here. The finding that morphine lowered body weight was expected. Previous studies 9'1°'16'2°'2s have shown that morphine can inhibit somatic development. In addition, studies using the opiate antagonist naltrexone have implicated endogenous opiates in the regulation of growth. Doses of naltrexone greater than 10 mg/kg daily from birth to day 21, which block the opiate receptor for 24 h/day, enhance somatic development, while lower doses inhibit development 29-31. Tyr-MIF-1 binds to its own non-opiate receptor and at higher doses, to the opiate receptor 22'26 where its
actions are complex: it can act as an antagonist when the receptor reserve is relatively low, but as an agonist in conditions of high receptor reserve-". Tyr-MIF-I alone, when administered prenatally, caused a significant increase in pup body weight on day 7. an antiopiate-like effect 24. In this study and in previous ones 23'24'27, Tyr-MIF-1 given in the postnatal period did not affect body weight. It is possible that a loss of the antiopiate effect of Tyr-MIF-I in the growing animal reflects the development of receptors and their coupling processes, resulting in a greater agonist-like activity of the peptide. Alternatively, these findings may be related to the Tyr-MIF-1 induced changes in the plasma concentration of growth hormone reported in adult rats 17. In contrast to the effects of morphine, two peptide agonists,/3-endorphin and morphiceptin, did not affect body weight when administered alone or in combination with Tyr-M1F-1 during the first week of life 27. In the present study, Tyr-MIF-1 not only failed to antagonize the effect of morphine, but slightly potentiated it. These effects of morphine and morphine + Tyr-MIF-I lasted at least 2 weeks beyond the neonatal period of treatment. Thus, although Tyr-MIF-1 can stimulate somatic development if given during the prenatal period 24, this antiopiate-like effect is no longer present if it is given postnatally when opiate receptors are more fully developed. As with body weight, Tyr-MIF-I neither affected eye opening by itself nor antagonized the delayed eye opening caused by morphine. The opiate antagonist naltrexone has been shown to accelerate eye opening when given at a higher dose (50 mg/kg) and for a longer time (days 0-21) than the test substances given in our study -~°. Similarly, MIF-1, an antiopiate peptide apparently without the opiate effects of Tyr-MIF-1, caused early eye opening in mice when given at a dose of 1 mg/kg on days 2-19 s. Thus, it is possible that Tyr-MIF-1 might have exerted an antiopiate effect if given for a longer time, but it is also possible that its opiate agonist actions would preclude such a change. Our group of pups receiving morphine on days 1-7 had a smaller proportion with eyes open on day 16 than did the control pups. In other studies 23'27, /3-endorphin, injected at doses and over the time identical to that of the present study, had no effect on eye opening. Pups pretreated with morphine were hypersensitive in the tail flick test on day 9. Tyr-MIF-1 did not antagonize this effect, nor did it affect the tail flick response when administered alone. In a previous study, neonatally administered /3-endorphin was shown to cause hypersensitivity in the tail flick test on day 9. However, this effect was antagonized by Tyr-MIF-127.
211
The present study, therefore, highlights the possible differences in the mechanisms of morphine-induced and /3-endorphin-induced hypersensitivity in the tail flick test and their differing susceptibilities to antagonism by the opiate-modulating peptide Tyr-M1F-1. The effects of morphine and Tyr-MIF-1 on the BBB transport system PTS-1 further illustrate the peptide's opiate-modulating properties. Although neonatally injected Tyr-MIF-1 did not have a significant effect by itself, it significantly potentiated morphine's effect of enhancing transporf of the labeled peptide out of the brain. Morphine previously had been shown to inhibit this rate of transport in adult mice when administered either acutely or chronically 5. However, that study measured transport 1 h after administration of morphine, whereas the present study measured it 2 weeks after administration of the drug had ended. Furthermore, paradoxical effects often occur with neonatal, as opposed to adult, administration of drugs. A long-term upregulation of transporters, induced by neonatal treatment, is implied. The presence of exogenous peptide or drug during a critical period could cause the development of an enhanced mechanism for removal, probably involving carrier-mediated transport through the BBB. The modulating site appears to be susceptible to both morphine and Tyr-M1F-1. In summary, neonatal administration of morphine can change, in a manner that Tyr-MIF-1 can in some cases potentiate, several developmental processes including somatic development, nociception, eye opening, and peptide transport across the blood-brain barrier.
Acknowled~q,ernents. This work was supported by the VA.
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