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Maintenance of beta-endorphin analgesia across age cohorts
Neurobiologyof Aging, Vol. 8, pp. 167-170.©PergamonJournals Ltd., 1987. Printed in the U.S.A.
0197-4580/87$3.00 + .00
Maintenance of Beta-Endorphin Analgesia Across Age Cohorts M A R I A - T E R E S A R O M E R O A N D R I C H A R D J. B O D N A W
Department of Psychology, Queens College, City University of New York, Flushing, N Y 11367 R e c e i v e d 23 July 1986 ROMERO, M.-T. AND R. J. BODNAR. Maintenance of beta-endorphin analgesia across age cohorts. NEUROBIOL AGING 8(2) 167-170, 1987.--Age-related decreases occur in analgesic responses following morphine, 2-deoxy-D-glucose, inescapable foot shock and cold-water swims. Decreased affinity and concentration of opiate receptors and levels of endogenous opioids are also observed. The present study evaluated the dose-dependent (0.1, 0.5, 1.0, 5.0/zg, ICV) and time-dependent (15, 30, 45, 60 min) properties of beta-endorphin analgesia on the jump test across three age cohorts of rats (8, 18 and 30 months of age). The different age cohorts failed to display differences in the magnitude of beta-endorphin analgesia across doses and times, except for a transient (30 min) decrease in the 30-month group following the 0.5 p.g dose. This maintenance of beta-endorphin analgesia across age cohorts stands in marked contrast to the age-related decrements in morphine and opiate-sensitive environmental analgesia and occurs despite decreased levels of beta-endorphin. These data are discussed in terms of differential alterations in opiate receptor subpopulations, and represent the first instance of maintained opioid analgesia across cohorts. Pain
Aging
Beta-endorphin analgesia
Jump test
OPIATE-MEDIATED analgesic responses have been shown recently to decline as a function of age, including morphine analgesia I l l , 22, 33], 2-deoxy-D-glucose analgesia [23], and inescapable forepaw shock analgesia [18]. The decreases in opiate analgesia and opiate-sensitive environmental analgesia as a function of age have typically been attributed to the corresponding decreases in opiate receptor and opioid peptide function. Decreases in the binding characteristics, affinity and concentrations of opiate receptors have been observed in regional and whole-brain assays [19, 27, 28] as well as decreases in levels of beta-endorphin, Leuenkephalin and Met-enkephalin [4, 5, 10, II, 15, 16, 29]. However, it is still unknown whether analgesia induced by administration of the endogenous opioids themselves is affected similarly by the aging process. Beta-endorphin, the C-terminal fragment of beta-lipotropin and primary active opioid component of the pro-opiomelanocortin system produces analgesia following central administration (see review [l]). The present study evaluated whether the analgesic effects of central administration of beta-endorphin differed across age cohorts as measured by the jump test which has previously been shown to be sensitive to age-related changes in analgesic processes [22,23].
Rats
Queens College Vivarium facility, and were not tested until they reached their respective cohort ages. All animals were maintained on a 12 hr light:12 hr dark cycle at ambient temperatures between 21 and 25°C with rat chow and water available ad lib. Before the beginning of testing, all animals in all groups were examined for presence of respiratory problems, abnormal body weight and tumour growth. Only animals that were free of these problems participated in the study. Female rats were chosen as experimental subjects to insure greater uniformity of body weights at the beginning of testing across cohorts: 8-month (mean=310; S E M = 9 g), 18-month (mean=323; SEM=16 g) and 30-month (mean=360; SEM=13 g). Before the onset of testing, all animals were administered chlorpromazine HC! (3 mg/ml normal saline/kg body weight, IP) 20 min before anesthetization with Ketamine HCI (100 mg/ml sterile water/kg body weight, IM). One stainless steel 22 gauge guide cannula (Plastic Products) was stereotaxically (Kopf) aimed so that its tip was positioned 0.3 mm above the lateral ventricle. With the incisor bar set at +5 mm, coordinates were 0.5 mm anterior to the bregma suture, 1.3 mm lateral to the sagittal suture and 3.6 mm from the top of the skull. The cannula was secured to three stainless steel anchor screws with dental acrylic. Ten days following surgery, jump thresholds of all groups were determined according to an ascending method of limits procedure [12]. Electric shocks were delivered by a 60-Hz constant current shock generator (BRS/LVE Model 901) and grid scrambler (Campden Instruments) through a 30×24 cm floor composed of 16 grids (0.3 cm diameter) spaced at 1.1 cm intervals. The jump threshold was defined in mA as the
METHOD Twenty-five female albino Sprague-Dawley rats (Sasco Laboratories) were represented across the following age cohorts: 8 (n=9), 18 (n=9) and 30 (n=7) months of age. Two age groups of animals were derived from the supplier: 2 and 11 months. All animals were housed individually in the
~Requests for reprints should be addressed to Richard J. Bodnar.
167
168
ROMERO AND BODNAR 15min
45 min
~.32
~.4~
.2 O
,
,
,
I .5
I 1
I 5
,
30min
i I .1
il,=i~l I 0 .1
.5
I 1
I 5
DOSE [ug]
FIG. I. Alterations in beta-endorphin analgesia on the jump test at 15, 30, 45 and 60 min after microinjection across beta-endorphin (0, 0.1,0.5, 1, 5/zg, ICV) doses and across age cohorts (8 months: closed circles; 18 months: closed triangles; 30 months: open squares). *Denotes significant (Dunn comparisons, p<0.05) agerelated changes in the magnitude of beta-endorphin analgesia relative to the 8month group. The standard errors of the mean for all data points varied between 0.008 and 0.043 mA.
lowest of two consecutive intensities that elicited simultaneous withdrawal of both hindpaws from the grids. Each trial began with the animal receiving a 300-msec foot shock at a current intensity of 0.1 mA. Subsequent shocks occurred at 10-sec intervals and were increased in equal 0.05 mA steps until the nociceptive threshold was determined. After each trial, the current intensity was reset to 0.1 mA for the next trial until 6 trials were completed. Following four days of stable baseline jump thresholds, each animal received the following five microinjections in ascending order: vehicle, and 0. I, 0.5, 1.0 and 5.0/xg doses of beta-endorphin (5/zl normal saline, Peninsula Laboratories). Infusions were done by hand using a Hamilton microsyringe with polyethylene tubing connecting a stainless steel 28 gauge internal cannula with the microsyringe. The rats were lightly restrained in a towel and the infusion was delivered at a rate of 1 tzl every 20 sec through the internal cannula which protruded 0.5 mm past the tip of the guide cannula. Jump thresholds were determined 15, 30, 45 and 60 min after injection. A minimum of four days elapsed between injection conditions to minimize tolerance effects, and the beta-endorphin doses were chosen on the basis of previous reports (e.g., [9,32]). Immediately following the completion of experimental testing, cannula placements of all animals were verified histologically as de-. scribed previously (e.g., [8]); all animals with appropriate cannula placements were included in the data analysis.
RESULTS Significant differences in jump thresholds were observed among the five microinjection conditions, F(4,88)=30.85, p<0.0001, and across the time course, F(3,66)=5.82, p <0.001, but not among groups, F(2,22) =0.01, nor for any of the interaction terms. Relative to corresponding vehicle values, significant analgesia was noted following the 0.1, 0.5, 1.0 and 5.0 tzg doses of beta-endorphin in all three groups. Figure 1 illustrates the failure of the different age cohorts to display differences in the magnitude of beta-endorphin
analgesia across doses and times, except for a transient decrease noted in the 30-month group relative to the 8-month group at 30 min following the 0.5 p.g dose of beta-endorphin (Dunn comparison, p<0.05).
DISCUSSION In contrast to previously-reported, age-related reductions m the opiate-mediated analgesic responses following morphine [21, 22, 33], 2-deoxy-D-glucose [23] and inescapable forepaw shock [18], the present data indicate that betaendorphin analgesia failed to be substantially affected across age cohorts between 8 and 30 months. Comparison of the present lack of age-related changes in central beta-endorphin analgesia with our previous [22] observation of age-related reductions in systemic morphine analgesia is noteworthy. Analgesia induced by 5 and 10 mg/kg doses of systemic morphine were reduced by 67c~ in 19-month and 24-month rats as compared to 9-month animals on the jump test. This reduction persisted across a 180-min time course, and reflected a 4-fold rightward shift in the systemic morphine analgesia dose-response curve. Comparisons between the lack of age-related central beta-endorphin analgesia changes with age-related shifts in systemic morphine analgesia raise the question that these effects are due to differences in injection routes. It should be noted that analgesic responses following both systemic and central morphine are similar on the basis of pharmacological (e.g., naloxone antagonism), neurophysiological (e.g., inhibition of dorsal horn nociceptive-sensitive neurons) and physiological (e.g., dorsal lateral funiculus transections) criteria (see review [6,14]). However, while central morphine and presumably betaendorphin appear to activate supraspinal analgesic mechanisms, systemic morphine activates both supraspinal and spinal systems which display some independence of function (see review [36]). It is conceivable that the agerelated effects upon systemic morphine analgesia are due to alterations in this spinal system; further studies are neces-
BETA-ENDORPHIN A N A L G E S I A AND AGING
169
sary to compare age-related changes in opiate analgesia elicited from spinal and supraspinal, localized injections. The failure of exogenously-administered beta-endorphin analgesia to vary as a function of age is surprising given the age-related reductions observed in endogenous betaendorphin content [4, 5, 10, 11, 15, 16, 29]. Such reductions might suggest a greater analgesic effect for beta-endorphin as a function of age due to receptor supersensitivity. Yet aging decreases the binding characteristics, affinity and concentrations of opiate receptors in regional and whole-brain assays [19, 27, 28]. In aged (26-month) female rats, dihydromorphine binding was reduced in brainstem, midbrain and thalamus relative to 5-month rats [28]. However, dihydromorphine allows for the characterization of mu receptors, but cannot distinguish effects upon other (delta, kappa, sigma and epsilon) subtypes [25, 26, 30, 35]. Since betaendorphin appears relatively selective for the epsilon receptor [2, 13, 17, 20], it is possible that aging produces selective changes in this receptor subtype. The age-related reductions in endogenous beta-endorphin can also result in larger proportionate increases in circulating levels when exogenous beta-endorphin is applied, and hence to hypothetically a greater analgesic response. However, aging increases the proportion of N-acetylated beta-endorphin [34], and acetylation decreases the opiate activity of endorphins [2,31]. Thus, the facilitating actions of proportional increases in beta-endorphin availability and the attenuating actions of greater age-related acetylation may cancel each other's effects and result in the failure to observe age-related effects. It should be noted that aging is not the only situation in
which such differential changes in opiate responses occur. Neonatal treatment with monosodium glutamate produces decreases in beta-endorphin [7, 9, 24] and reduces morphine, but not beta-endorphin analgesia [3, 7, 9]. Neonatal monosodium glutamate treatment also potentiates the analgesic response to the delta receptor agonist, D-Ala2-DLeuS-enkephalin [9] and selectively increases the number of delta, but not mu receptors [37]. These data suggest that the differential opiate analgesic effects induced by neonatal monosodium glutamate may be due to corresponding differential changes in opiate receptor subtypes. This possibility may hold for age-related effects as well. Differences in opiate analgesic potency as a function of age have potential clinical relevance. Therefore, it is important to ascertain whether analgesia induced by other opiate receptor subtype agonists (e.g., D-Ala2-D-LeuS-enkephalin: delta; ketocyclazocine, dynorphin: kappa) are decreased, unaffected or even increased as a function of age, and then to correlate such differences with the binding characteristics of opiate receptor subtypes. These issues were not addressed in the present report since this study was the last of a current series of experiments (e.g., [21,22]). Further work is necessary to investigate these relationships.
ACKNOWLEDGEMENTS This research was supported by NIA Grant AG-04425, NIH BRSG RR07064 and PSC/CUNY Grants 6-65193 and 6-66351 to R.J.B.
REFERENCES
1. Akil, H., S. J. Watson, E. Young, M. E. Lewis, H. Khachaturian and J. M. Walker. Endogenous opioids: biology and function. Annu Rev Neurosci 7: 223-255, 1984. 2. Akil, H., E. Young, S. J. Watson and D. H. Coy. Opiate binding properties of naturally occurring N- and C-terminus modified beta-endorphins. Peptides 2: 28%292, 1981. 3. Badillo-Martinez, D., A. L. Kirchgessner, P. D. Butler and R. J. Bodnar. Monosodium glutamate and analgesia induced by morphine. Neuropharmacoh)gy 23:1141-1149, 1984. 4. Barden, N., A. Dupont, F. Labile, Y. Merand, D. Roulou, H. Vaudry and J. R. Boissier. Age-dependent changes in the beta-endorphin content of discrete rat brain nuclei. Bra#~ Res 208: 209-212, 1981. 5. Barnea, A., G. Cho and J. C. Porter. A reduction in the concentration of immunoreactive corticotropin, melanotropin and lipotropin in the brain of the aged rat. Brain Res 232: 345-353, 1982. 6. Basbaum, A. I. and H. L. Fields. Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. Annu Rev Neurosci 7: 30%338, 1984. 7. Bodnar, R. J., G. W. Abrams, E. A. Zimmerman, D. T. Krieger, G. Nicholson and J. S. Kizer. Neonatal monosodium glutamate: effects upon analgesic responsivity and immunocytochemical ACTH//3-1ipotropin. Neuroendocrinology 30: 280-284, 1980. 8. Bodnar, R. J., A. L. Kirchgessner, G. Nilaver, J. Mulhern and E. A. Zimmerman. Intraventricular capsaicin: alterations in analgesic responsivity without depletion of substance P. Neuroscience 7: 631-638, 1982. 9. Bodnar, R. J., T. Portzline and G. Nilaver. Differential alterations in opioid analgesia following neonatal monosodium glutamate treatment. Brain Res Bull 15: 299-305, 1985.
10. Dorsa, D. M., E. R. Smith and J. M. Davidson. Immunoreactive-fl-endorphin and LHRH levels in the brains of aged male rats with impaired sex behavior. Neurobiol Aging 5: 115-120, 1984. I I. Dupont, A., P. Savard, Y. Merand, F. Labile and J. R. Boissier. Age-related changes in central nervous system enkephalins and substance P. Life Sci 29: 2317-2322, 1981. 12. Evans, W. O. A new technique for the investigation of some analgesic drugs on a reflexive behavior in the rat. Psychopharmacologia 2: 318-325, 1961. 13. Ferrar, P., R. Houghten and C. H. Li. /3-Endorphin: characteristics of binding sites in the rat brain. Biochem Biophys Res Commun 89: 786--792, 1979. 14. Fields, H. L. and A. I. Basbaum. Brainstem control of spinal pain transmission neurons. Annu Rev Physiol 40:217-248, 1978. 15. Forman, L. J., W. E. Sonntag, D. A. Van Vugt and J. Meites. Immunoreactive beta-endorphin in the plasma, pituitary and hypothalamus of young and old male rats. Neurobiol Aging 2: 281-284, 1981. 16. Gambert, S. R., T. L. Garthwaite, C. H. Pontzer and T. C. Hagen. Age-related changes in central nervous system betaendorphin and ACTH. Neuroendocrinology 31: 252-255, 1980. 17. Goodman, R. R., R. A. Houghten and G. W. Pasternak. Autoradiography of 3H-fl-endorphin binding in brain. Brain Res 288: 334-337, 1983. 18. Hamm, R. J. and J.~S. Knisely. Environmentally-induced analgesia: an age-related decline in an endogenous opioid system. J Gerontol 40: 268-274, 1985. 19. Hess, G. D., J. A. Joseph and G. S. Roth. Effect of age on sensitivity to pain and brain opiate receptors. Neurobiol Aging 2: 4%55, 1981.
170 20. Houghten. R. A., N. Johnson and G. W. Pasternak. ZH-/3endorphin binding in rat brain. J Neurosci 4: 2460-2465, 1984. 21. Kavaliers, M., M. Hirst and G. C. Teskey. Aging, opioid analgesia and the pineal gland. Life Sci 32: 227%2287, 1983. 22. Kramer, E. and R. J. Bodnar. Age-related decrements in morphine analgesia: A parametric analysis. Neurobiol Aging 7: 185-191, 1986. 23. Kramer, E., E. S. Sperber and R. J. Bodnar. Age-related decrements in the analgesic and hyperphagic responses to 2deoxy-D-glucose. Physiol Behav 35: 92%934, 1985. 24. Krieger, D. T., A. S. Liotta, G. Nicholson and J. S. Kizer. Brain ACTH and endorphin reduced in rats with MSG arcuate nucleus lesions. Nature 278: 562-563, 1979. 25. Lord, J. A. H., A. A. Waterfleld, J. Hughes and H. W. Kosterlitz. Endogenous opioid peptides: multiple agonists and receptors. Nature 253: 495-499, 1977. 26. Martin, W. R., C. G. Eades, J. Thompson, R. E. Huppler and P. E. Gilbert. The effects of morphine- and nalorphine-like drugs in the nondependent and morphine dependent chronic spinal dog. J Pharmacol Exp Ther 197: 517-522, 1976. 27. Messing, R. B., B. J. Vasquez, B. Samaniego, R. A. Jensen, J. L. Martinez and J. L. McGaugh. Alterations in dihydromorphine binding in cerebral hemispheres of aged male rats. J Neurochem 36: 784--790, 1981. 28. Messing, R. B., B. J. Vasquez, V. R. Spiehler, J. L. Martinez, R. A. Jensen, H. Rigter and J. McGaugh. 3H-dihydromorphine binding in the brain regions of young and aged rats. Life Sei 26: 921-927, 1980.
ROMERO AND BODNAR 29. Missale, C., S. Govani, L. Croce, A. Bosio, P. F. Spano and M. Trabucchi. Changes of beta-endorpbin and Met-enkephalin content in the hypothalamus-pituitary axis induced by aging../ Neurochem 40: 20-24, 1983. 30. Schulz, R., M. Wuster and A. Herz. Pharmacological characterization of the epsilon opiate receptor. J Pharmaeol Exp Ther 216: 786-792, 1981. 31. Smyth, D. G., D. E. Massey, S. Zakarian and M. D. A. Finnie. Endorphins are stored in biologically active and inactive forms: isolation ofalpha-N-acetyl peptides. Nature 279: 252-254, 1979. 32. Tseng, L. F., S. S. Cheng and J. M. Fujimoto. Inhibition of tail-flick and shaking responses by intrathecal and intraventricular D-Ala2-D-LeuS-enkephalin and beta-endorphin in anesthetized rats. J Pharmaeol Exp Ther 224:51-54, 1983. 33. Webster, G. W., L. Shuster and B. E. Eleftheriou. Morphine analgesia in mice of different ages. Exp Aging Res 2: 221-223, 1976. 34. Wilkinson, C. W. and D. M. Dorsa. The effects of aging on molecular forms of beta- and gamma-endorphins in rat hypothalamus. Neuroendoerinology 43: 124--131, 1986. 35. Wuster, M., R. Schulz and A. Herz. The direction of opioid agonists toward mu, delta and epsilon receptors in the vas deferens of the mouse and the rat. Lift, Sci 27: 163-170, 1980. 36. Yaksh, T. L. Spinal opiate analgesia: characterization and principles of action. Pain 11: 293-346. 1981. 37. Young, E., J. Olney and H. Akil. Selective alterations of opiate receptor sub-types in monosodium glutamate-treated rats..I Neurochem 40: 1558-1564, 1983.
0197-4580/87$3.00 + .00
Maintenance of Beta-Endorphin Analgesia Across Age Cohorts M A R I A - T E R E S A R O M E R O A N D R I C H A R D J. B O D N A W
Department of Psychology, Queens College, City University of New York, Flushing, N Y 11367 R e c e i v e d 23 July 1986 ROMERO, M.-T. AND R. J. BODNAR. Maintenance of beta-endorphin analgesia across age cohorts. NEUROBIOL AGING 8(2) 167-170, 1987.--Age-related decreases occur in analgesic responses following morphine, 2-deoxy-D-glucose, inescapable foot shock and cold-water swims. Decreased affinity and concentration of opiate receptors and levels of endogenous opioids are also observed. The present study evaluated the dose-dependent (0.1, 0.5, 1.0, 5.0/zg, ICV) and time-dependent (15, 30, 45, 60 min) properties of beta-endorphin analgesia on the jump test across three age cohorts of rats (8, 18 and 30 months of age). The different age cohorts failed to display differences in the magnitude of beta-endorphin analgesia across doses and times, except for a transient (30 min) decrease in the 30-month group following the 0.5 p.g dose. This maintenance of beta-endorphin analgesia across age cohorts stands in marked contrast to the age-related decrements in morphine and opiate-sensitive environmental analgesia and occurs despite decreased levels of beta-endorphin. These data are discussed in terms of differential alterations in opiate receptor subpopulations, and represent the first instance of maintained opioid analgesia across cohorts. Pain
Aging
Beta-endorphin analgesia
Jump test
OPIATE-MEDIATED analgesic responses have been shown recently to decline as a function of age, including morphine analgesia I l l , 22, 33], 2-deoxy-D-glucose analgesia [23], and inescapable forepaw shock analgesia [18]. The decreases in opiate analgesia and opiate-sensitive environmental analgesia as a function of age have typically been attributed to the corresponding decreases in opiate receptor and opioid peptide function. Decreases in the binding characteristics, affinity and concentrations of opiate receptors have been observed in regional and whole-brain assays [19, 27, 28] as well as decreases in levels of beta-endorphin, Leuenkephalin and Met-enkephalin [4, 5, 10, II, 15, 16, 29]. However, it is still unknown whether analgesia induced by administration of the endogenous opioids themselves is affected similarly by the aging process. Beta-endorphin, the C-terminal fragment of beta-lipotropin and primary active opioid component of the pro-opiomelanocortin system produces analgesia following central administration (see review [l]). The present study evaluated whether the analgesic effects of central administration of beta-endorphin differed across age cohorts as measured by the jump test which has previously been shown to be sensitive to age-related changes in analgesic processes [22,23].
Rats
Queens College Vivarium facility, and were not tested until they reached their respective cohort ages. All animals were maintained on a 12 hr light:12 hr dark cycle at ambient temperatures between 21 and 25°C with rat chow and water available ad lib. Before the beginning of testing, all animals in all groups were examined for presence of respiratory problems, abnormal body weight and tumour growth. Only animals that were free of these problems participated in the study. Female rats were chosen as experimental subjects to insure greater uniformity of body weights at the beginning of testing across cohorts: 8-month (mean=310; S E M = 9 g), 18-month (mean=323; SEM=16 g) and 30-month (mean=360; SEM=13 g). Before the onset of testing, all animals were administered chlorpromazine HC! (3 mg/ml normal saline/kg body weight, IP) 20 min before anesthetization with Ketamine HCI (100 mg/ml sterile water/kg body weight, IM). One stainless steel 22 gauge guide cannula (Plastic Products) was stereotaxically (Kopf) aimed so that its tip was positioned 0.3 mm above the lateral ventricle. With the incisor bar set at +5 mm, coordinates were 0.5 mm anterior to the bregma suture, 1.3 mm lateral to the sagittal suture and 3.6 mm from the top of the skull. The cannula was secured to three stainless steel anchor screws with dental acrylic. Ten days following surgery, jump thresholds of all groups were determined according to an ascending method of limits procedure [12]. Electric shocks were delivered by a 60-Hz constant current shock generator (BRS/LVE Model 901) and grid scrambler (Campden Instruments) through a 30×24 cm floor composed of 16 grids (0.3 cm diameter) spaced at 1.1 cm intervals. The jump threshold was defined in mA as the
METHOD Twenty-five female albino Sprague-Dawley rats (Sasco Laboratories) were represented across the following age cohorts: 8 (n=9), 18 (n=9) and 30 (n=7) months of age. Two age groups of animals were derived from the supplier: 2 and 11 months. All animals were housed individually in the
~Requests for reprints should be addressed to Richard J. Bodnar.
167
168
ROMERO AND BODNAR 15min
45 min
~.32
~.4~
.2 O
,
,
,
I .5
I 1
I 5
,
30min
i I .1
il,=i~l I 0 .1
.5
I 1
I 5
DOSE [ug]
FIG. I. Alterations in beta-endorphin analgesia on the jump test at 15, 30, 45 and 60 min after microinjection across beta-endorphin (0, 0.1,0.5, 1, 5/zg, ICV) doses and across age cohorts (8 months: closed circles; 18 months: closed triangles; 30 months: open squares). *Denotes significant (Dunn comparisons, p<0.05) agerelated changes in the magnitude of beta-endorphin analgesia relative to the 8month group. The standard errors of the mean for all data points varied between 0.008 and 0.043 mA.
lowest of two consecutive intensities that elicited simultaneous withdrawal of both hindpaws from the grids. Each trial began with the animal receiving a 300-msec foot shock at a current intensity of 0.1 mA. Subsequent shocks occurred at 10-sec intervals and were increased in equal 0.05 mA steps until the nociceptive threshold was determined. After each trial, the current intensity was reset to 0.1 mA for the next trial until 6 trials were completed. Following four days of stable baseline jump thresholds, each animal received the following five microinjections in ascending order: vehicle, and 0. I, 0.5, 1.0 and 5.0/xg doses of beta-endorphin (5/zl normal saline, Peninsula Laboratories). Infusions were done by hand using a Hamilton microsyringe with polyethylene tubing connecting a stainless steel 28 gauge internal cannula with the microsyringe. The rats were lightly restrained in a towel and the infusion was delivered at a rate of 1 tzl every 20 sec through the internal cannula which protruded 0.5 mm past the tip of the guide cannula. Jump thresholds were determined 15, 30, 45 and 60 min after injection. A minimum of four days elapsed between injection conditions to minimize tolerance effects, and the beta-endorphin doses were chosen on the basis of previous reports (e.g., [9,32]). Immediately following the completion of experimental testing, cannula placements of all animals were verified histologically as de-. scribed previously (e.g., [8]); all animals with appropriate cannula placements were included in the data analysis.
RESULTS Significant differences in jump thresholds were observed among the five microinjection conditions, F(4,88)=30.85, p<0.0001, and across the time course, F(3,66)=5.82, p <0.001, but not among groups, F(2,22) =0.01, nor for any of the interaction terms. Relative to corresponding vehicle values, significant analgesia was noted following the 0.1, 0.5, 1.0 and 5.0 tzg doses of beta-endorphin in all three groups. Figure 1 illustrates the failure of the different age cohorts to display differences in the magnitude of beta-endorphin
analgesia across doses and times, except for a transient decrease noted in the 30-month group relative to the 8-month group at 30 min following the 0.5 p.g dose of beta-endorphin (Dunn comparison, p<0.05).
DISCUSSION In contrast to previously-reported, age-related reductions m the opiate-mediated analgesic responses following morphine [21, 22, 33], 2-deoxy-D-glucose [23] and inescapable forepaw shock [18], the present data indicate that betaendorphin analgesia failed to be substantially affected across age cohorts between 8 and 30 months. Comparison of the present lack of age-related changes in central beta-endorphin analgesia with our previous [22] observation of age-related reductions in systemic morphine analgesia is noteworthy. Analgesia induced by 5 and 10 mg/kg doses of systemic morphine were reduced by 67c~ in 19-month and 24-month rats as compared to 9-month animals on the jump test. This reduction persisted across a 180-min time course, and reflected a 4-fold rightward shift in the systemic morphine analgesia dose-response curve. Comparisons between the lack of age-related central beta-endorphin analgesia changes with age-related shifts in systemic morphine analgesia raise the question that these effects are due to differences in injection routes. It should be noted that analgesic responses following both systemic and central morphine are similar on the basis of pharmacological (e.g., naloxone antagonism), neurophysiological (e.g., inhibition of dorsal horn nociceptive-sensitive neurons) and physiological (e.g., dorsal lateral funiculus transections) criteria (see review [6,14]). However, while central morphine and presumably betaendorphin appear to activate supraspinal analgesic mechanisms, systemic morphine activates both supraspinal and spinal systems which display some independence of function (see review [36]). It is conceivable that the agerelated effects upon systemic morphine analgesia are due to alterations in this spinal system; further studies are neces-
BETA-ENDORPHIN A N A L G E S I A AND AGING
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sary to compare age-related changes in opiate analgesia elicited from spinal and supraspinal, localized injections. The failure of exogenously-administered beta-endorphin analgesia to vary as a function of age is surprising given the age-related reductions observed in endogenous betaendorphin content [4, 5, 10, 11, 15, 16, 29]. Such reductions might suggest a greater analgesic effect for beta-endorphin as a function of age due to receptor supersensitivity. Yet aging decreases the binding characteristics, affinity and concentrations of opiate receptors in regional and whole-brain assays [19, 27, 28]. In aged (26-month) female rats, dihydromorphine binding was reduced in brainstem, midbrain and thalamus relative to 5-month rats [28]. However, dihydromorphine allows for the characterization of mu receptors, but cannot distinguish effects upon other (delta, kappa, sigma and epsilon) subtypes [25, 26, 30, 35]. Since betaendorphin appears relatively selective for the epsilon receptor [2, 13, 17, 20], it is possible that aging produces selective changes in this receptor subtype. The age-related reductions in endogenous beta-endorphin can also result in larger proportionate increases in circulating levels when exogenous beta-endorphin is applied, and hence to hypothetically a greater analgesic response. However, aging increases the proportion of N-acetylated beta-endorphin [34], and acetylation decreases the opiate activity of endorphins [2,31]. Thus, the facilitating actions of proportional increases in beta-endorphin availability and the attenuating actions of greater age-related acetylation may cancel each other's effects and result in the failure to observe age-related effects. It should be noted that aging is not the only situation in
which such differential changes in opiate responses occur. Neonatal treatment with monosodium glutamate produces decreases in beta-endorphin [7, 9, 24] and reduces morphine, but not beta-endorphin analgesia [3, 7, 9]. Neonatal monosodium glutamate treatment also potentiates the analgesic response to the delta receptor agonist, D-Ala2-DLeuS-enkephalin [9] and selectively increases the number of delta, but not mu receptors [37]. These data suggest that the differential opiate analgesic effects induced by neonatal monosodium glutamate may be due to corresponding differential changes in opiate receptor subtypes. This possibility may hold for age-related effects as well. Differences in opiate analgesic potency as a function of age have potential clinical relevance. Therefore, it is important to ascertain whether analgesia induced by other opiate receptor subtype agonists (e.g., D-Ala2-D-LeuS-enkephalin: delta; ketocyclazocine, dynorphin: kappa) are decreased, unaffected or even increased as a function of age, and then to correlate such differences with the binding characteristics of opiate receptor subtypes. These issues were not addressed in the present report since this study was the last of a current series of experiments (e.g., [21,22]). Further work is necessary to investigate these relationships.
ACKNOWLEDGEMENTS This research was supported by NIA Grant AG-04425, NIH BRSG RR07064 and PSC/CUNY Grants 6-65193 and 6-66351 to R.J.B.
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